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 Table of Contents  
Year : 2022  |  Volume : 11  |  Issue : 2  |  Page : 190-198

A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis

1 Department of Immunology, National School of Biological Sciences, National Polytechnic Institute, Mexico City, México
2 Laboratory of Tuberculosis, National Institute of Forestry, Agricultural and Livestock Research, Mexico City, México

Date of Submission19-Feb-2022
Date of Decision20-May-2022
Date of Acceptance28-May-2022
Date of Web Publication14-Jun-2022
Date of Print Publicaton14-Jun-2022

Correspondence Address:
Oscar Rojas-Espinosa
Department of Immunology, National School of Biological Sciences, National Polytechnic Institute, Carpio y Plan de Ayala S/n, Colonia Santo Tomás, 11410 Mexico City
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_71_22

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Background: Bovine tuberculosis (bTB) is still a prominent threat to animal health; lacking an efficient vaccine, other than BCG to get rid of tuberculosis, the most effective way for this is culling and slaughtering the infected animals. There are several cellular, serological, and molecular tests for the diagnosis of the disease but the most practical one at the field level is the double skin testing with bovine and aviary tuberculins. This is not a very specific test but is sensitive enough to identify most diseased animals; adjunct practical tests are desirable to strengthen the utility of skin tests. All lymphoid and myeloid cells participate, in diverse grades, in the immune response to tuberculosis with neutrophils playing an unintended pathologic role. The study aimed to investigate the response of neutrophils to agents present in the sera of tuberculous cows. Methods: We have developed a neutrophil-based test (N BT) to identify diseased cows within a herd suspected of having tuberculosis; a positive N BT correlates with a positive double skin test. In this test, healthy neutrophils are incubated with the sera of healthy or tuberculous cows for 3 and 6 h, and the nuclear morphologic changes are recorded and analyzed. Results: Sera from tuberculous but not from healthy cows induce nuclear alterations including pyknosis, swelling, apoptosis, and sometimes NETosis, in healthy neutrophils, and CFP 10 and ESAT 6 participate in the phenomenon. Conclusion: We propose the N BT as an auxiliary tool for substantiating the diagnosis of bTB reinforcing the PPD test outcome to help decide whether or not a cow should be sacrificed.

Keywords: Bovine tuberculosis, ESAT6, Mycobacterium bovis, neutrophils, PPD

How to cite this article:
Rojas-Espinosa O, Beristain-Cornelio G, Santillán-Flores MA, Arce-Paredes P, Islas-Trujillo S, Rivero-Silva M&. A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis. Int J Mycobacteriol 2022;11:190-8

How to cite this URL:
Rojas-Espinosa O, Beristain-Cornelio G, Santillán-Flores MA, Arce-Paredes P, Islas-Trujillo S, Rivero-Silva M&. A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis. Int J Mycobacteriol [serial online] 2022 [cited 2022 Jul 6];11:190-8. Available from: https://www.ijmyco.org/text.asp?2022/11/2/190/347530

  Introduction Top

Bovine tuberculosis (bTB) is a disease of cattle and other domestic and wild mammals, but also of people.[1] It is caused by Mycobacterium bovis (M. bovis) whose principal entry route is the upper respiratory tract, although it may also penetrate the skin through dermal abrasions, and the intestinal mucosa by ingestion of contaminated food or water. Newborn cattle may also become infected through the ingestion of contaminated milk or colostrum.[2],[3]

Bovine pulmonary tuberculosis evolves as a chronic granulomatous disease affecting the lungs and the regional lymph nodes[4] as it happens in human tuberculosis.[5] Here, the infection may remain subclinical for months or years until the overt manifestation of the disease occurs; frequently the lesions disseminate affecting other parts of the lung or other organs. Latent tuberculosis (latency) has been confirmed by positive interferon- or tuberculin skin tests in asymptomatic animals.[3],[6]

Histopathologically, bTB very much resembles human tuberculosis,[4] and many findings in human tuberculosis are taken as surrogates in bTB.[7] Once M. bovis enters the lung and reaches the alveolar spaces, it is phagocytosed by resident alveolar macrophages and dendritic cells which become activated and start confronting the microorganism releasing in so doing, toxic and inflammatory mediators, including reactive oxygen intermediaries and cytokines such as interleukin-1b (IL), IL-6, IL-12, and tumor necrosis factor (TNF), among others. TNF and IL-17 (produced by local innate lymphoid T cells-3, (ILC-3), stimulate endothelial cells to release neutrophil-attracting chemokines[8],[9] and inflammatory mediators that ease the entry of lymphocytes and blood-borne macrophages to the inflammation site. Soon, alveolar dendritic cells (ADC) that have started managing the microorganism migrate to the draining lymph node to initiate the anti-mycobacterial adaptive immune response. In the lymph node, ADC presents their antigenic cargo to naive lymphocytes which under the influence of diverse cytokines differentiate into the functional phenotypes Th1, Th2, Th17, Th9, Th22, Treg, and cytotoxic T-cells.[10] Although all cell phenotypes participate in anti-tuberculosis immunity, some of them are particularly relevant, namely lymphocytes Th1, Th17, and Tc (CD8+). Instructed by dendritic cells, lymphocytes will proliferate and release cytokines (the ones corresponding to the activated T-cell phenotype) and enter the blood and lymphatic circulation to disseminate throughout the body, eventually passing by the developing tuberculous granuloma where macrophages, neutrophils, mycobacteria, and mycobacterial antigens are present. Here, the already primed T cells will accumulate contributing to the granuloma maturation. Infiltrating T cells will promote the activation of alveolar and immigrant macrophages, making them more efficient bacterial killers. Macrophages in turn will activate newly arrived lymphocytes inducing their proliferation and the release of more cytokines and chemokines, including neutrophil-recruiting IL-17 and the chemokines CXCL8 (IL-8), CXCL1, CXCL2, and CXCL5, among others.[11] At the same time, follicular dendritic cells present in the germinal centers of the lymphoid follicles will trap soluble mycobacterial antigens and immune complexes which upon recognition by B follicular lymphocytes will activate these cells to start the production of antimycobacterial antibodies,[12] thus closing the circle of antituberculosis immunity.

Under this scenario, if the resulting cell-mediated immune response (CMIR) is strong enough, it may deter the granuloma development, limiting bacilli proliferation, and the infection will completely disappear, or remain in the latent state, with no symptoms or signs of disease. If, on the contrary, the CMIR is not strong enough, then bacilli will grow leading to a bacilliferous granuloma prone to disruption and bacillary dissemination. Furthermore, a sustained anti-mycobacterial inflammatory response will eventually lead to cell-mediated hypersensitivity with progressive tissue damage including necrosis and liquefaction, the origin of the cavitary lesions of tuberculosis.[13]

After the infection is established, and prompted by mycobacterial products, by TNFα from macrophages and IL-17 from ILC-3, neutrophils will soon arrive in large numbers at the infection site.[9] In an in vivo study with 13 cows intranasally infected with 2 × 107 M. bovis, Cassidy et al.[14] found macrophages and giant cells containing neutrophil debris and acid-fast bacilli (AFB) as early as 3 days' post-infection in the alveolar spaces. On day 14, multifocal neutrophil aggregates with AFB were observed surrounded by a mantle of macrophages. By day 21, extensive central necrosis of neutrophils and macrophages was observed producing an amorphous mass surrounded by a mantle of intact and degenerated neutrophils, giant cells, and lymphocytes. Similar lesions but with larger necrotic centers were observed by days 28 and 42 post-infection. The role of neutrophils in the anti-tuberculous immunity probably ends here, capturing and fighting M. bovis (with only partial success) before dying by apoptosis, necrosis, and/or NETosis. An indirect microbicidal activity of neutrophils occurs through the release of bacilli-containing apoptotic bodies and microvesicles[15],[16] which are then ingested by macrophages to take on the bacteria-killing.

Simultaneously to the granuloma development mycobacterial antigens are released into the surrounding tissue, including peripheral blood where they may have effects on the circulating cells. This has been demonstrated in human tuberculosis where sera from patients with active pulmonary tuberculosis induced marked apoptotic changes in the nuclear morphology of healthy neutrophils.[17] The precise nature of the mycobacterial antigens responsible for the changes was not identified in that work but ESAT-6 was a strong candidate.

Due to the close similarities in the immunopathology of human and bTB, we envisaged that a similar effect of sera from tuberculous cows on the nuclear morphology of neutrophils might occur. Thus, sera from tuberculous (PPD+) and nontuberculous (PPD–) cows were tested on human neutrophil monolayers, and the results are presented in this article.

  Methods Top


Unless otherwise stated, most chemicals were purchased from Sigma-Aldrich, Mexico. Recombinant ESAT-6 and CFP-10 were from Prospec, Rehovot 7670308, Israel; NBP1-05468 Anti-ESAT-6 antibody was from Novus Biologicals, CO 80112, USA.

Serum samples

Cow sera were obtained from blood collected from the tail vein, by personnel of INIFAP involved in the National Surveillance Programme for bTB. Cattle were Holstein milk cows from the State of Mexico, México, a region with a low prevalence (≈0.5%) of tuberculosis. Tuberculous cows were identified based on clinical symptomatology, the comparative intradermal test (bovine and aviary PPD), and the interferon-gamma releasing test in some cases.

Neutrophil monolayers

Human neutrophils were isolated from venous blood taken from the arm of healthy donors that signed their informed consent (Project CEI-ENCB, SH-007-2020). Cells were isolated by centrifugation of 3.0 ml of heparinized blood on 3.0 ml of Polymorphprep (Axis-Shield, Oslo Norway) for 60 min at 1500 RPM and 25°C. The neutrophils layer (second from top) was collected, washed once with Phosphate-buffered saline, pH 7.4, added 0.1% glucose (PBSG), suspended in 1 ml of PBSG, and counted in a hemocytometer. The working cell suspension was adjusted to 1 × 106 cells per ml. Neutrophil monolayers were prepared by seeding 40 μl of the cell suspension (40 × 103) on circular areas (0.8 cm Ø) demarcated on sterile glass slides. The charged slides were placed into Petri dishes and the dishes were incubated for 30 min at 37°C in a humid atmosphere under 5% CO2 to allow the adhesion of neutrophils to the glass slides.

Treatment of monolayers

With sera

Petri dishes were retrieved and the supernatant fluid on the neutrophil monolayers was carefully removed and replaced with 40 μl of undiluted, healthy, or tuberculous, cow sera. Then, monolayers were incubated for periods ranging from 0 to 6 h. At the end of the incubation time, monolayers were recovered, and the supernatant fluid was removed and substituted with 50 μl of 2% paraformaldehyde for 10 min. Finally, after rinsing with distilled water, monolayers were stained with the Hoechst reagent for 2 min, rinsed, and mounted with Vecta shield for microscope examination. In some cases, monolayers were also post-stained for 1 min with Iris Fuchsia, a red fluorescent stain.

With mycobacterial extracts

Rinsed monolayers were incubated with 50 μl of sera from healthy cows to which several amounts of Mycobacterium tuberculosis extract in 10 μl-aliquots were added. M. tuberculosis extracts were prepared by sonication, centrifugation, and filtration through 0.2 μm nitrocellulose membranes; the amount of protein in the extracts was adjusted to 1 mg per ml. The incubation time ranged from 1 to 6 h at 37°C/5%CO2.

With recombinant proteins ESAT-6, CFP-10, P-38, and 85B

Rinsed monolayers were incubated for 3 h with 50 μl of sera from healthy cows to which 1.0 μg of the proteins were added. To eliminate the effect of LPS this experiment was performed in the presence of 1 μg of polymyxin b.

With Quantiferon components

Tubes with Quantiferon components (nill, mitogen, antigens) were treated with 1 ml of distilled water to dissolve contents. Then, 10 μl of each solution were added to the neutrophil monolayers in 50 μl of serum from healthy cows, and the monolayers were incubated for 3 h at 37°C/5%CO2.

Antimycobacterial antibodies

Antimycobacterial antibodies were measured by a standard enzyme-linked immunosorbent assay (ELISA) including coating of the wells (NUNC) with 5 μg of whole M. tuberculosis protein extracts in 0.01 M bicarbonate buffer, pH 8.6, for 18 h, washing thrice with 0.01M phosphate buffer, pH 7.4, blocking with 3% skim milk in carbonate buffer for 60 min, the addition of sera diluted 1:100 in PBS, incubation at 37°C for 2 h, washing thrice with PBS, the addition of peroxidase-rabbit anti-bovine immunoglobulins diluted 1:1000 in PBS, incubation for 1 h at 37°C, washing thrice with PBS, the addition of chromogen-substrate solution (3 mg ortho phenylenediamine and 10 μl of 30% hydrogen peroxide in 10 ml of PBS), incubation for 20 min, the addition of 4N sulfuric acid, and registration of absorbances at 492 nm in an ELISA reader (Multiskan Plus, Thermo Fisher, USA).

Apoptosis assay

Apoptosis was evaluated by using the Annexin V FITC Assay kit of Cayman Chemical (Ann Arbor, Michigan, USA) following the manufacturers' indications. The analysis was made by flow cytometry (488/527 nm for FITC and 655/739 for PI) in a FACSCalibur® instrument (BD Biosciences, San José CA, USA).

Analysis of results

Neutrophils were examined for nuclear changes, including pyknosis, swelling, apoptosis, and NETosis. The statistical analysis when needed is mentioned in the corresponding results.

  Results Top

Sera from PPD + but not from PPD − cows induce nuclear changes in human neutrophils

Initially, undiluted sera from ten tuberculous (PPD+) or ten healthy (PPD−) cows were incubated in the presence of neutrophils for several periods (37°C/5% CO2). Although changes started to be noticed by 60 min of incubation, they were evident at 120 min and were maximal at 180 min in the PPD+ group. On the contrary, changes were almost absent in the PPD negative group even at the longest incubation time. [Figure 1] shows the results at 3 h incubation.
Figure 1: Sera from tuberculous cows (TBC-1 to 4) variably induce nuclear changes in human neutrophils. The most frequent findings were pyknotic (P), swollen (S), and pre-apoptotic (A) nuclei. Some sera induced lesser but still noticeable changes (serum TBC-4). Sera from healthy cows (HC-1 to 4) did not induce nuclear changes in neutrophils. This is a representative result of over 40 sera analyzed. Bars are 20 μm scales. Hoechst stain, ×40

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Pyknosis, nuclei swelling, and apoptosis were the most frequent changes induced by tuberculous cow sera on human neutrophils

[Figure 2] depicts the percent of cells with normal, pyknotic, or swollen nuclei after 2 h, 4 h, or 6 h incubation with a pool of ten sera from tuberculous (PPD+, IGRA+) cows. Pyknotic and swollen nuclei were observed at 4 h incubation but cells with swollen nuclei were predominant at 6 h incubation.
Figure 2: Sera from tuberculous cows induce progressive nuclear changes in neutrophils. Pyknotic (P), swollen (S) and pre-apoptotic (A) nuclei were the most frequent alterations provoked by tuberculous sera on human neutrophils. Neutrophils with normal nuclear morphology (N) predominated at early times of incubation (0 and 1 h). Nuclear changes increased proportionally to the incubation time, being swollen (S) and pre-apoptotic (A) nuclei the predominant end alteration. Hoechst stain, ×40

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Neutrophil's morphologic changes include pyknosis, nuclear swelling, and apoptosis

The most prominent changes induced by the sera from the PPD+ group included pyknosis, swelling, and apoptosis and they appeared proportional to the time of incubation. Once again, changes were noticed from 2 h of incubation [Figure 3].
Figure 3: Nuclear changes in neutrophils were observed at 3 and 6 h incubation. The results with three TBC and three HC are illustrated. Tuberculous sera that induced apoptotic changes in neutrophils at 3 h, did not worsen the changes at 6 h incubation. Most neutrophils incubated with sera of TBC showed swollen preapoptotic-like nuclei. Notice that neutrophils incubated with sera from healthy cows did not show significant changes at 6 h incubation. Bars are 20 μm scales. Hoechst and Iris fuchsia stains, ×40. TBC: Tuberculous cows, HC: Healthy cows

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Anti-mycobacterial antibodies are not an inducer of nuclear changes

As expected, sera from tuberculous cows had higher levels of antimycobacterial antibodies than sera from healthy cows (P = 0. 0335) but the level of antibodies did not correlate with the ability of sera to induce nuclear changes in neutrophils. [Figure 4] illustrates the results of an experiment with 10 sera randomly taken from healthy or tuberculous cows.
Figure 4: Anti-M. bovis antibodies in the sera of tuberculous cows. Sera from TBC contain higher levels of anti-M. bovis antibodies than sera from HC (P = 0.0335). however, there was no significant correlation between the levels of antibody and the ability of sera to induce nuclear changes in neutrophils (Pearson r = 0.29, P = 0.442, n = 10). TBC: Tuberculous v, HC: Healthy cows

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Mycobacterial antigens induce nuclear changes in neutrophils

Suspecting the presence of soluble mycobacterial antigens in the serum of tuberculous cows and their potential role as change-inducers in neutrophils, neutrophil monolayers were incubated for 3 h (37°C and 5% CO2) in the presence of variable amounts of soluble antigens extracted from M. bovis. As observed in [Figure 5], amounts as small as 0.5 μg can induce clear nuclear changes in neutrophils and increase with higher antigen concentrations. Changes are similar to the ones induced by tuberculous-cow sera.
Figure 5: Soluble Mycobacterium bovis antigens (SMBA) induce nuclear changes in neutrophils. 40,000 cells were incubated, for 3 h, in the presence of variable amounts of SMBA (4.0 to 0.25 ug). Clear changes started to be noticed from 0.5 μg and increased proportionally to the antigen concentration. The graph corresponds to only this experiment and refers to the number of neutrophils with obvious nuclear changes per field (the illustrated) at the different antigen concentrations. Three separated experiments were performed with similar results. Hoechst stain, ×40.

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Activation of human neutrophils with ESAT-6 and CFP-10

Diverse mycobacterial antigens may function as change-inducers in neutrophils, but ESAT-6 and CFP-10 are particularly relevant. To prove that mycobacterial CFP-10 and ESAT-6 are inducers of nuclear changes, these proteins (recombinant) were incubated for 3 and 6 h with human neutrophils. CFP-10 and ESAT-6 are conserved proteins within the Mycobacterium tuberculosis complex (MTC) although they have also been found outside the MTC.[18] Both proteins induced nuclear changes in neutrophils that increased with the incubation time [Figure 6]. Other recombinant proteins tested, P38 and 85B, did not induce meaningful changes in neutrophils.
Figure 6: ESAT-6 and CFP-10 induce nuclear changes in neutrophils. Neutrophil monolayers were incubated with PBSG, CFP-10, or ESAT-6 for 3 and 6 h. PBSG did not alter the nuclear morphology of neutrophils at any incubation time. ESAT-6 more than CFP-10 induced nuclear changes that increased from 3 to 6 h incubation. Changes were very variate and included pyknosis, nuclear swelling (apoptosis), and DNA release. Hoechst´s stain, ×40. PBSG: Phosphate-buffered saline, pH 7.4, added 0.1% glucose

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ESAT-6 induces nuclear changes in neutrophils at very low concentrations

Nuclear changes in neutrophils were induced with amounts of recombinant ESAT-6 as small as 300 ng/test [Figure 7]. This result emphasizes the biological effect of this protein on peripheral leukocytes.
Figure 7: ESAT-6 induces nuclear changes in neutrophils at trace concentrations. Forty thousand neutrophils were incubated in the presence of increasing amounts (300 ng to 10 μg) of recombinant ESAT-6 for 3 h at 37°C%–5%CO2. Changes started to be noticed with the lowest concentration of ESAT-6 used and increased proportionally to the amount of ESAT-6. The most noticeable changes (encircled) were pyknosis (p), chromatin swelling (s), and apoptosis (a). Worth of notice is that a small number of neutrophils remained unchanged (n, normal) even at the highest concentration of ESAT-6 used. Bars are 20 ted to be noticed

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To corroborate that ESAT-6 participates in the phenomenon, three bovine inducer-sera were preincubated for 60 min with 0.0, 1.0, 0.5, or 0.250 μg of the anti-ESAT-6 monoclonal antibody, and then assessed on the neutrophil's monolayers. The nuclear changes-inducer effect was almost completely abolished when sera were incubated with 1.0 μg of the neutralizing antibody and its neutralizing effect diminished proportionally to the antibody concentration. No effect was observed when neutrophils were incubated with sera from healthy cows [Figure 8].
Figure 8: Neutralization of ESAT-6 in serum. Forty microliters of tuberculous cow sera were incubated for 60 min with 0, 1.0, 0.5, or 0.25 μg of anti-ESAT-6 antibody, and then tested on neutrophil-monolayers for 3 h. The results of 2 experiments (I and II) are shown: untreated tuberculous sera (a) induced over 90% nuclear alterations, while 1.0 μg of antibody blocked in 80% the capacity of sera to induce nuclear changes (b). Lesser amounts of antibody were proportionally less inhibitory (c and d). Neutrophils (Neu) in the presence of healthy sera showed no more than 10% alterations (image not shown). Hoechst stain, ×40

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The main nuclear changes in neutrophils induced by tuberculous-cow sera are suggestive of apoptosis

The late effect of tuberculous cow sera on neutrophils is apoptosis as suggested by the Annexin stain and the morphologic nuclear appearance. However, apoptosis does not seem to be complete as nuclear fragmentation and apoptotic bodies are not observed even at 6 h of incubation, and the phenomenon could be more suggestive of early apoptosis, or another form of cell death that we tentatively call “contained NETosis,” a phenomenon that we are further investigating. [Figure 9] illustrates this result.
Figure 9: The late effect of bovine tuberculous sera on the nuclear morphology of neutrophils is apoptosis. (a and b) show the flow cytometer distribution of neutrophils incubated for 6 h in the presence of serum from a HC (a), or serum from a TBC (b). These are representative results. (c) illustrates the global percent of apoptotic neutrophils incubated in the presence of 20 sera from HC or 20 sera from TBC. (d) Shows the apoptotic appearance of neutrophils incubated in the presence of two TBC sera (Left: Hoechst and iris fuchsia stains, right: negative images of Hoechst stain), ×40. Horizontal lines are 20 μm scales. TBC: Tuberculous cows, HC: Healthy cows, One way ANOVA with post-Tukey test

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  Discussion Top

bTB is an important disease of cattle that not only implies economic losses but also poses a threat to human and veterinary welfare (Macedo-Couto et al., 2019).[19] Despite the many attempts to prevent the disease by vaccination, except for Bacille Calmette–Guérin (BCG), no new vaccines have been developed or approved. BCG vaccination, however, has not been accepted in developed countries because of its low effectiveness and the concern that it may sensitize the vaccinated cows lowering the diagnostic value of the intradermal PPD skin test (IST). Thus, so far, the only way to eradicate the disease is culling and slaughtering the animals that result positive in the IST, but the reassessment of BCG vaccination with improved vaccines that do not affect the PPD reactivity is being considered.[20],[21],[22]

Neutrophils in tuberculosis are key participants not only because of their antimicrobial activity but also because they play a role in the pathogenesis of the disease. It is known that neutrophils circulate in an activated state in human tuberculosis,[23] and they surely do so in bTB. During active tuberculosis, many physiological changes occur in the granuloma and these changes are reflected in the blood highly likely affecting the structure and function of circulating cells, namely leukocytes, including neutrophils the predominant leukocyte population in blood.

An activated state of circulating neutrophils will reflect what is happening in the granuloma, thus, detecting these activated cells may help to uncover an ongoing disease in a cow under clinical suspicion of tuberculosis. A positive “neutrophil test” may help to confirm the clinical diagnosis, reinforcing the veterinarian's decision on the fate of the cow.

In this communication, we have developed a “neutrophil-based test (N-BT)” and have confirmed its diagnostic value as sera from PPD+ but not PPD-cows induce nuclear alterations in human neutrophils. The test is a simple one, but extreme care is required for its execution as neutrophils are extremely sensitive cells that immediately react to any irritating stimuli. In our methodology, we use a one-step separation process with no hypotonic treatment, this allows recovering neutrophil populations with over 95% purity and viability as determined by the Trypan blue exclusion test. For practical reasons human neutrophils, instead of bovine neutrophils, are used as the target cells but comparing the response of human and bovine neutrophils is an activity that will be done shortly. We have also looked for the factor in the serum of tuberculous cows responsible for the changes induced on neutrophils. After discarding the participation of antimycobacterial antibodies and IL-8, we found that soluble mycobacterial antigens and particularly ESAT-6 are deeply involved in the process; blockage of ESAT 6 with a specific antibody, led to almost complete inhibition of the nuclear changes' inducer ability of tuberculous sera. In addition, recombinant ESAT6 showed similar activity to mycobacterial extracts and tuberculous sera to induce nuclear changes in neutrophils. Furthermore, ESAT-6 has been invested with regulatory effects on all cells of the immune system[24] and has been reported to be able to induce the production of IL-8 in lung epithelial cells, thus participating in the granuloma formation.[25] It has also been described as a leukocidin causing calcium influx, necrosis, and formation of extracellular traps in neutrophils.[26] It also affects antigen presentation by macrophages, possibly in conjunction with CFP-10.[27] In a pathology as complex as bTB, it is expected that ESAT-6 may play a variety of roles.

  Conclusion Top

Fine-tuning of the changes observed in this report is necessary to define if they correspond to apoptosis, necrosis, or any other form of cell death (“contained NETosis”); this analysis is currently underway in our laboratory. Finally, we hope that someone else will try this N-BT to validate its potential usefulness as an adjunct diagnostic tool in bTB. The test, however, does not seem to be specific for tuberculosis as sera from cows with paratuberculosis behave in an analogous manner (these results are not presented in this paper because very few serum samples from cows with paratuberculosis, brucellosis, viral diarrhea, and infectious rhinotracheitis have been analyzed).

Limitation of study

The number of tuberculous and healthy cows should be increased, as should the number of cows with other pathologies. This will be done as soon as the COVID-19 pandemia is over.

Ethical clearance

Research Committee of the National Institute of Forestry, agriculture, and Livestock Research, Palo Alto, Ciudad de Mexico (INIFAP: 20 01 2019), and Committee of Ethics on Research of the National School of Biological Sciences, National Polytechnic Institute (CEI ENCB: 29-01-2021).

Financial support and sponsorship

This study received financial support from Secretaría de Investigación y Posgrado (SIP: 20180099) del Instituto Politécnico Nacional, México. Authors are fellowship-holders of CONACYT (O R-E, G B-C), COFAA (O R-E, P A-P), EDI (O R-E), and EDD (P A-P).

Conflicts of interest

There are no conflicts of interest.

Further methodological details will be given on request.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]


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