Clinical and immunological study of specific Mycobacterium bovis-BCG transfer factor in lambs

1. The prepared M. bovis-BCG Transfer factors was safe and have no side effects on recipient lambs.
2. The prepared transfer factor induced cell mediated immunity detected by DTH in recipient lambs.

3.  There was a strong relationship between serum level of IFN-γ, IL17 and MIF in the specific M. bovis-BCGTF recipient lambs.
4.  The prepared TF improve the cytokine production (IL17. IFNγ and MIF).
5. Further investigation are needed to determine the physicochemical characterization of TF extracted from various sources.

Clinical and immunological study of specific Mycobacterium bovis-BCG transfer factor in lambs

Elaf Abdulqader Nayyef ¹, Mawlood Abbas Ali Al-Graibawi²

¹Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Fallujah, Al-Anbar, ²Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq

aaylaf10@gmail.com, https://orcid.org/0000-0002-7218-2435, 07817314857

algraibawi_57@yahoo.com, https://orcid.org/0000-0003-2218-2813, 07707166772

Abstract

This study was designed to prepare and evaluate the specific Mycobacterium bovis (M. bovis) Bacillus Calmette-Guerin (BCG) transfer factor (TF) from spleen cells of two lambs vaccinated subcutaneously S/C with BCG vaccine twice 2 weeks’ intervals, similarly one lamb injected with normal saline used as a donor of non-specific TF. The lambs were clinically examined at zero time and along 3 days post each immunization, then, delayed type hypersensitivity (DTH) was checked post 4 weeks. TF was obtained from spleen cells of the lambs, inspected for sterility; safety.TF was evaluated in 4 groups (five in each) of lambs 3-4 months old. The first group (recipient) was injected S/C with 2 ml of specific BCG-TF, similarly the 2nd, 3rd and 4th groups were injected with non-specific TF, BCG and normal saline respectively. The clinical signs and DTH test were checked as described above, only the specific TF recipient and BCG vaccinated lambs gave a positive DTH test. The serum concentration of inerlukin-17 (IL-17), interferon-gamma (IFN-γ) and migration inhibitory factor (MIF) cytokines were measured by Enzyme-linked immunosorbent assay (ELISA), which revealed a highly significant increase in the TF recipient lambs in comparison with the control and vaccinated groups. It concluded that the prepared specific M. bovis-BCG transfer factor was effective to induce cell mediated immunity via DTH and increasing the serum concentration of cytokines in recipient lambs.

Keywords: Transfer factor, Immunotherapy, IL-17, IFN-γ, MIF Cytokine

دراسة سریریة، مناعیة للعامل الناقل الخاص بلقاح السلالمحضر فی الحملان

إیلاف عبدالقادر نایف¹ومولود عباس علی الغریباوی²

¹ فرع الطب الباطنی والوقائی البیطری، کلیة الطب البیطری، جامعة الفلوجة، الأنبار، فرع الطب الباطنی والوقائی البیطری، کلیة الطب البیطری، جامعة بغداد، بغداد، العراق

الخلاصة

صممت هذه الدراسة لتحضیر وتقییم العامل الناقل الخاص بلقاح السل من طحال الحملان الملقحة. تم تحصین اثنان من الحملان تحت الجلد مرتین فی فترة أسبوعین بلقاح السل واستخدمت کمانحة للعامل الناقل الخاص بجراثیم السل وحقن الحمل الثالث بمحلول ملحی عادی کمانح للعامل الناقل غیر الخاص بجراثیم السل. فحصت الحملان سریریا فی ساعة الصفر وبعد کل تحصین عن ظهور أی علامات سریریة لمدة ثلاثة أیام. بعد أربعة أسابیع فحصت الحملان بوساطة اختبار حساسیة الجلد. استخلص العامل الناقل من الخلایا الطحالیة للحملان، وفحص عن العقم والسلامة، قیمت کفاءته المناعیة فی أربع مجامیع من الحملان بعمر ٣-٤ شهر (خمسة لکل مجموعة)، حقنت المجموعة الأولى (المستلمة) تحت الجلد باثنان ملیلیتر من العامل الناقل الخاص بجراثیم السل، وبنفس الطریقة حقنت المجموعة الثانیة والثالثة والرابعة بالعامل الناقل غیر الخاص بجراثیم السل ولقاح السل وبمحلول ملحی عادی على التوالی. فحصت الحیوانات سریریا وأجری اختبار حساسیة الجلد المتأخربعد اربعة اسابیع من التلقیح. اظهرت الحملان المستلمة للعامل الناقل الخاص والحملان الملقحة بلقاح السل نتیجة موجبة لاختبار حساسیة الجلد المتأخر. قیست تراکیز السایتوکینات (انتیرفیرون غاما و انترلوکین - ١٧ و عامل تثبیط هجرة البلعمیات) فی المصل باستخدام اختبار الالیزا المناعی التنافسی، وأظهرت زیادة معنویة بتراکیزها فی المجموعة المستلمة للعامل الناقل الخاص بلقاح السل مقارنة مع المجامیع الاخرى. بینت هذه الدراسة کفاءة العامل الناقل الخاص بلقاح السل لأحداث مناعة خلویة من خلال اختبار حساسیة الجلد المتأخر وزیادة تراکیز السایتوکینات فی الحملان المستلمة.

Introduction

Transfer factor, also called dialyzable leukocyte extract (DLE) or ‘Lawrence transfer factor, firstly described in 1949 by Lawrence (1), it is very small molecular weight (lower than 5,000 Daltons), that able to transfer cellular immune responses from a sensitized host (donor) to non-sensitized one (recipient), and protect them against pathogens, its immunological activity was evaluated against many bacterial, viral, parasitic and fungal diseases (2). The general term transfer factor denotes numerous highly active biological agents that have a general immune correctors properties (immunomodulatory, immunizing and immunosubstituting), and are obtained from various biological sources such as, blood leucocytes, spleen, lymph node, yolks of bird eggs, colostrums of mammals (3,4).

Since cellular immunity is essential for controlling of infectious disease, autoimmune disorders, cancer, immunodeficiency and allergies, TF is an immune regulator which activates cellular immunity, stimulates cytokine synthesis and regulates immune function and can be applied for the treatment and prevention of these cases (5). Its essential mechanism of action has been proposed at the level of cellular immune responses as it induces synthesis of IL 1, IL17, MIF and IFN-γcytokines (6). Cytokines are a low molecular weight regulatory peptide that participates in both types of immunity (innate and adaptive), which play a vital role in the infectious and chronic disease (7). They are important immune system communication mediators and are important for host protection against pathogens (8). Leukocyte’s ability to react to a microbial stimulus is affected by cytokines, which may cause systemic or localized immune response by transmitting signals through cellular receptors (9).

Bacillus Calmette-Guerin is an attenuated vaccine against Mycobacterium tuberculosis (M. tuberculosis) currently; it is the only authorized and widely used vaccine in the world for prevention of TB in human (10). It prepared from attenuated of M. bovis strain, it is the most effective vaccine to induce immunity against the three dangerous mycobacterial agents: (M. tuberculosis, M. lepraeand M. bovis) (11). This vaccine awarded a significant protection to experimentallyinfected lambsthat measured through decreasing bacterial load and size of gross lesion (12).

Reviewing the available recent studies, there is no report concerning the preparation and evaluation of specific M. bovis- BCG TF, in addition to analysis of the cytokines IL17, MIF and IFN gamma in the recipient lambs. This study aimed to prepare specific M. bovis- BCG transfer factor and evaluate its clinical and some immunological activity in lambs.

Materials and methods

Experimental animals

A total number of 23 Awassi lambs, 3-4 months old, and 6 albino rabbits (1.2-2kg) used in the current study, were housed in the animal farm of the College of Veterinary Medicine, University of Baghdad, they were fed, alfalfa and commercial assorted pellets, barley, bran and clean water. The animals were adapted for two weeks before starting the experiments to ensure disease-free and treated with albendazole and ivermectin, nasal swabs and fecal samples were collected for diagnosis of bacterial and parasitic infection (13). This study was approved by The Committee of Ethics Research of the Department of Internal and Preventive Medicine, College of Veterinary Medicine, University of Baghdad, Ministry of High Education and Scientific Research-Iraq (No. 1300/ 11-10-2020).

Immunization and clinical examination

Three Awassi lambs (3-4 months old) were used to prepare and extract the TF, two lambs, vaccinated twice S/C with 0.5 ml of BCG (Pasteur and Danish, obtained from the Iraqi Ministry of Health) vaccine,two weeks’ intervals as donor of specific TF (14), and the third lamb injected with normal saline used as a donor of non-specific TF. Lambs were monitored daily for clinical signs observations (temperature, respiratory rate and heart rate) 3 days post each immunization. After 4 weeks’ success of immunization was confirmed by DTH-skin test.

Delayed type hypersensitivity-skin test

Four weeks post the immunization; the left flank area of the lamb was clipped and shaved carefully,and divided into two portions, the firstone was injected I.D with 0.1 mL of normal saline as a control, while the second one were injected with 0.1 mL of purified protein derivative (PPD). The thickness of skin reaction was assayed by the ruler at 72 hours after inoculation (15), TF was extracted from spleen cell of the lambs.

Transfer factor extraction

Two weeks post the 2nd immunization, TF was obtained from the splenocyte of the vaccinated and control lambs as described previously (16),the spleen were removed aseptically, chopped and subjected to mechanical disintegration and grunt with clean sterile sand in sterile mortar and cell extract were suspended with sterile normal saline, the number cells was counted by hemacytometer, then it was adjusted at 5x10⁸ cell/ml, andsubjected to twelve times for freezing at -20℃ and thawing(at 37℃ to disrupt the cells . The disrupted cells were centrifuged with a high speed cold centrifuge at 15000 rpm for 40 min. to eliminate cellular debris. Supernatant was filtrated by 0.45 and 0.22 Millipore filter, then ultra-filtered via Amicon filter (Merck Amicon-Ultra Centrifugal filter) to obtain the lysate containing transfer factor (4). The lysate was sterilized via a 0.22 Millipore filter, and kept at -20°C for later use. The Sterility was confirmed by culturing of some drops of TF on blood agar and incubated at 37°C for 48 hours (16). Safety was confirmed by injection of TF in rabbits.

First experiment

To estimate the safety and biological activity of the prepared TF to transfer DTH-reaction, six rabbits were divided equally in to 2 groups, the first groups injectedintra-peritoneal (I.P) with 2 mL equivalent to 5*10⁸ cells/mL of specific TF and the second group injected I.P with 2 mL normal saline and monitored daily for any clinical signs and checked for cellular immunity by DTH test one week later (15).

Second experiment

Twenty Awassi lambs (3-4 months old) were divided equally and randomly into four groups to estimate the immunological activity of the prepared specific transfer factor, the first group was injected twice two weeks’ interval S/C with 2 ml (equivalent to 5x10⁸cells /ml) of specific BCG-TF, similarly the 2^nd, 3^rdand 4^thgroups were injected with non-specific TF (2 ml equivalent to 5x10⁸cells /ml), BCG 0.5ml and normal saline 2 ml, respectively. Clinical examination was carried out to all animals at zero time and post injection, which includes heart rate, rectal temperature and respiratory rate, blood sample were collected for 3 times post 2^ndimmunization for serum gathering. All the lambs were examined by DTH-skin test as described above at the 4^thweek post immunization (15).

Estimation of cytokines level

Blood samples were obtained from jugular vein of all lambs at1^st, 3^rdand 4^thweeks post 2^ndimmunization, sera were isolated by centrifugation at 2000 rpm for 15 min (17). The cytokine concentrations of IL17, IFN-γ and MIF were estimated according to the manufactures guidelines of the sheep ELISA Kit (MyBioSource USA).

Statistical analysis

Statistical analysiswas carried out to detect the statistical variation between the groups by using two-way analysis of variance (ANOVA), the data were presented as mean and standard error.

Result

Clinical examination post immunization

Prior to immunization, all lambs in the four groups, specific TF, BCG, non-specific TF and control showed normal clinical parameters (body temperature respiratory and heart rates) Post immunization, only the BCG group revealed a significant increase at P≤0.05 in these parameters, the peaks of at 24 hours then returned to normal ranges at 72 hours after immunization (Figures 1-3).Small local swelling was detected through palpation at the injection site of BCG vaccine, the peak of increased in body temperature, respiratory rate and heart rates were 40.2±0.10ºC, 32.2±1.19 rate/min, and 81.4±0.97 rate/min respectively.

Figure 1: Body temperature in specific TF, BCG, non-specific TF and control lambs.

Figure 2: Respiratory rates/min. in specific TF, BCG, non-specific TF and control lambs.

Figure 3: Heart rates/minute in specific TF, BCG, non-specific TF and control lambs.

Delayed-type hypersensitivity test

The results DTH test showed positive skin reaction in the specific TF recipient lambs and BCG vaccinated lambs only (Table 1), characterized by indurations, erythema and increase in skin thickness in the left flank of lambs after 24 hours. and became at peak after 48 hours and persisted to72 hours, while thecontrol and non-specific TF recipient groups showed no skin reaction at injection sites. The thickness of skin in specific TF recipient lambs and BCG vaccinated lambs were >5mm.

Table 1: skin reaction in specific TF, BCG, non-specific TF and control lambs

Interferon gamma

The IFN-γcytokine level revealed a significant increase in the specific TF recipient and BCG groups in the comparison with non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization (Figure 4).

Figure 4: Serum IFN γ level in specific TF, BCG, non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization.

Interleukin-17

The IL17 level revealed a significant increase at only in the specific TF recipient group in the comparison BCG, non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization (Figure 5).

Figure 5: Serumlevel of IL17 in specific TF, BCG, non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization.

Macrophage migration inhibitory factors

The MIF level revealed a significant increase only in the specific TF recipient group in the comparison BCG, non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization(Figure 6).

Figure 6: Serum level of MIF in specific TF, BCG, non-specific TF and control lambs at 1^st, 3^rdand 4^thweek post booster immunization.

Discussion

The results of immunization of lambs with BCG vaccine showed a significant increase in the clinical parameters (body temperature respiratory and heart rates) and small local swelling was detected through palpation at the injection site of BCG vaccine, this may be due to its immunological and inflammatory reactions as described formerly byTizard (18). Similar finding was reportedpreviously byAbdulrazaq and Al-Graibawi(19) they reported that rabbits injected with M. bovis-BCG revealed slight depression, swelling palpated at injection site by 96 hours and a slight increase in body temperature 38.2-40.1ºC. While the pilot study showed that the immunization of the rabbits with the prepared transfer factor was safe and did not induce systemic any adverse reactions, this may be due to the small size of the TF, which did not induce any inflammatory reaction (4).

 Bacillus Calmette-Guerin is an attenuated vaccine prepared from attenuated strain of M. bovis, it is used to induce immunity against TB, and it is the most effective vaccine against all three major mycobacterial agents: M.tuberculosis, M. bovis and M. leprae(11). BCG is a safe vaccine have been already used in numerous kinds of animals TB hosts such as, cattle, goats, white tailed deer, badgers, possums, and African buffalo (10,12).

The positive DTH-skin test reactivity in the specific TF recipient and BCG vaccinated lambs post I/D injection of PPD in comparison with no reaction in control and nonspecific TF recipient lambs in the present study, this may be due to cellular immune responses transferred by specific TF and that induced by BCG vaccine (4,18,19).Cellular immunity playsa pivotal role in the defense of host against diseases; therefore, the assessment of CMI is important for evaluation of host defense against diseases. Various tests have been developed to estimate cell mediated immunity, among these tests, DTH- skin test, phagocytic index and cytokines assay (18). The traditional method for measuring the response of BCG vaccination by detecting the DTH-skin test reaction to intra-dermal PPD is tuberculin test (20).

The current study revealed significantly increased in the cytokines level (IFN-γ, IL17 and MIF) in specific TF recipient lambs compared to other lambs of experiment, this may be due to activation and differentiation of cells that responsible for these cytokines production (21). This results agreements with Whillefordetal., (23) who reported that post inoculation of mice with serum derived TF the IFN-γ increased 77 fold (7.8 to 597pg/ml) at 24 hours. TF have the ability to regulate the immune responses, which can cause the synthesis of IFN-γ from NK cells (5).

IL-17 and IFN-γ and MIF are soluble, tiny molecular weight glycopeptides and polypeptides released by a various cell types that have enhancing or suppressive impacts on cellular proliferation, motility differentiation, and activation. Most of them are not constitutively produced, but are secreted in response to activation by infectious agents or them by products like: endotoxin, mechanical injuries, inflammatory mediators, and cytokines themselves (7,8).

Transfer factor has the ability to regulate the innate immune responses, which induce the synthesis of IFN-γ from NK cells (5). The innate and adaptive immune responses play a vital role in the prevention and control the mortality caused by COVID -19 infections (22,24). For this reason, the TF is recommended as adjuvant therapeutic agent in the prevention and control symptoms associated with COVID-19, this recommendation depending on several studies concerning the using of TF in the SARS-CoV-2 infection (25).

Conclusions

The current study demonstrated the efficacy of prepared specific M. bovis-BCG transfer factor to induce cell mediated immunity via DTH and increasing the serum concentration IL-17, IFN-γ and MIF in recipient lambs.

Acknowledgment

 Authors would like to thanks the College of Veterinary Medicine, University of Fallujah,Al-Anbar,College of Veterinary Medicine, University of Baghdad, Baghdad, and the College of Veterinary Medicine in supporting of current research and University of Mosul, Mosul for accepting this article, Iraq.

Conflict of interest

The authors declare that there are no conflicts of interest regarding the publication of this manuscript.