Current status of vaccine research, development, and challenges of vaccines for Mycoplasma gallisepticum.

Mycoplasma gallisepticum (MG) is an important avian pathogen that causes significant economic losses in the poultry industry. Surprisingly, the limited protection and adverse reactions caused by the vaccines, including live vaccines, bacterin-based (killed) vaccines, and recombinant viral vaccines is still a major concern. Mycoplasma gallisepticum strains vary in infectivity and virulence and infection may sometimes unapparent and goes undetected. Although extensive research has been carried out on the biology of this pathogen, information is lacking about the type of immune response that confers protection and selection of appropriate protective antigens and adjuvants. Regardless of numerous efforts focused on the development of safe and effective vaccine for the control of MG, the use of modern DNA vaccine technology selected in silico approaches for the use of conserved recombinant proteins may be a better choice for the preparation of novel effective vaccines. More research is needed to characterize and elucidate MG products modulating MG-host interactions. These products could be used as a reference for the preparation and development of vaccines to control MG infections in poultry flocks.

Introduction

Mycoplasma gallisepticum (MG) is an avian pathogen, belongs to the class mollicutes and characterized by a special feature, the lack of cell wall (Razin and Herrmann, 1998; Lu et al., 2017). The pathogen primarily infects chickens and turkeys (Ley, 2003), but also some other birds, such as house and wild finches (Ley et al., 1996; Dhondt et al., 2014) and geese (Benoina et al., 1988). It is well documented that MG is the causative agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys (Chin et al., 1991; Cecchini et al., 2007; Wijesurendra et al., 2015; Beaudet et al., 2017; Kanci et al., 2018). The disease is often slowly developed and accompanied with severe inflammation in the respiratory tract of birds (Harry and Yoder, 1990). The pathogen often remains undetected in flocks and caused latent infections (Evan et al., 2005). Previous reports showed that factors such as high feeding density, heat and/cold stresses, excessive ammonia, accumulation of feces, fouling of the chicken house, excessive temperature differences, and sudden changes in climate can contribute to the spread and outbreak of disease (Hochachka and Dhondt, 2000; Ley, 2003). Mycoplasma gallisepticum infection caused major economic losses in terms of reduced weight gain, egg production and hatchability, downgrading carcass quality, and the infected birds become susceptible to other diseases (Beaudet et al., 2017; Ishfaq et al., 2019a). Mycoplasma gallisepticum is a continuing problem in poultry and efforts to prevent the losses in commercial flocks particularly in layer and breeders including bacterin-based vaccines, killed vaccines, and live vaccines. These efforts have been successful up to some extent in reducing the severity of respiratory diseases, maintaining constant egg production, controlling excess vaccine reactions, and reducing horizontal and vertical transmission (Butcher, 2002). Among these vaccines, some vaccines are semi-virulent, often have adverse effect, and provide only partial or transient immunity (Jacob et al., 2014, 2015; Peebles et al., 2014, 2015). Moreover, in view of the increasing antimicrobial resistance and decreasing effectiveness of antibiotics in controlling MG infection, and alarmingly, exacerbation of disease is often associated with vaccine improvement, the need for effective and novel vaccines has become even more necessary (Kleven et al., 1984; Whithear, 1996; Kleven, 2008). Table 1 shows the currently available vaccines against MG infection. Numerous studies so far, focused on live-attenuated vaccine, killed vaccines, bacterin-based, or recombinant proteins (Whithear, 1996; Hussein et al., 2007; Rabie and Amin Girh, 2020). This review assesses and summarizes the description of the problem, challenges, and vaccines under development or in use and highlighted developments that may be better for the preparation of effective vaccines in the future.

Table 1

List of commercial vaccines available in the market.

S. no.	Name	Vaccine type and strain	Manufacturer
1	AviPro104 MG BACTERIN	Bacterin, Strain R	Lohmann Animal Health International, United States.
2	Mycoplasma gallisepticum vaccines	Live-attenuated vaccine, Strain R, Strain F-36	Shandong Lvdu Biosciences and Technology Co., Ltd. Some other companies in different cities of China.
3	VAXSAFE MG VACCINE	Live-attenuated vaccine, Strain ts-11	Bioproperties Pty LTD Australia
4	Mycoplasma Gallisepticum Vaccine	Live-attenuated vaccine, Strain ts-11	Boehringer Ingelheim Animal Health Business Unit
5	Nobilis MG 6/85	Live vaccineStrain 6/85	MSD Animal Health PHILIPPINES, INC.
6	CEVAC MG F	Live-attenuated vaccine, Strain F	Ceva Santé Animale, United Kingdom.
7	Mycoplasma Gallisepticum Vaccine (TS-11)	Live vaccineStrain ts-11	MERIAL, INC. (POULTRY BIOLOGICS), GAINESVILLE, GA, 30,503, United States.
8	MG-Bac Vaccine	Bacterin	United States.
9	Vaxsafe MG (Strain TS-11)	Live-attenuated vaccine, Strain ts-11	Rhone Ma Malaysia Sdn Bhd.

Challenges

Despite extensive research on the pathology of MG, there are several challenges that hinder the triumph of vaccines to prevent MG infection. Among these challenges are immune status of host animals, colonization of MG in host respiratory tract, presence of other respiratory pathogens, phase or antigenic variation that lead to immune escape from the host immune system and transmission of infection in the flocks (Levisohn et al., 1995; Staley and Bonneaud, 2015; Beaudet et al., 2017; Beaudet et al., 2019). Adhesion plays a pivotal role in the colonization and pathogenesis of Mycoplasmas infection (Henderson and Jensen, 2006). Mycoplasma gallisepticum adhere to the host cells with the help of a terminal bleb, which in turn damage cell architecture and the surface layer of epithelial cells lose its cilia, leading to increased chances of other secondary infections (Uppal and Chu, 1977; Bredt, 1979). Up till now, several adhesion proteins have been reported to involve in adhesion and colonization of MG. For instance, Glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (Alvarez et al., 2003; Dumke et al., 2011), PvpA (Yogev et al., 1994), GapA (Goh et al., 1998), and Crm A or mgc3 (Yoshida et al., 2000; Indiková et al., 2013), MGC2 (Hnatow et al., 1998), pyruvate dehydrogenase-α and pyruvate dehydrogenase-β (Qi et al., 2018), and OsmC-like protein MG1142 (Jenkins et al., 2007). The infection caused the release of catarrhal exudate from the goblet cells and lead to mucosal thickness of the respiratory epithelium and has the ability to invade various host tissues and cells such are chicken erythrocytes, fibroblasts, and HeLa-229 cells. This invasive capability contributes to the dissemination of MG to various distant sites in the host body and the successful establishment of persistent and recurrent infection (Baseman et al., 1995; Mohammed et al., 2007; Majumder et al., 2014; Rosales et al., 2017; Ishfaq et al., 2019b, 2019c). However, factors such as strain of MG, infected dose, passage, and host immunity also greatly affects the establishment of MG infection. Moreover, studies demonstrated that the host often failed to recognize MG because of its antigenic/phase variation of membrane surface proteins, which are associated with evasion from the host immune system leading to infection (Gorton and Geary, 1997; Markham et al., 1998; Glew et al., 2000; Mazin et al., 2014). Mycoplasma gallisepticum modulate host immune system through activation of toll-like receptors (TLR) including TLR-2/TLR-4/TLR-6, nod-like receptors, and NF-κB pathway (Takeda et al., 2002; Gaunson et al., 2006; Majumder et al., 2014; Li et al., 2019; Chen et al., 2020). Increased number of CD4+ and CD8+ lymphocytes were found in chicken tracheal mucosa after 1 wk of infection (Gaunson et al., 2000, 2006). Another study reported that MG infection increased the expression of CXCL13, CXCL14, lymphotactin, RANTES, IL-1β, MIP-1β, and IFN-γ gene in chicken trachea (Mohammed et al., 2007; Majumder and Silbart, 2016). Similarly, Beaudet et al., reported immune dysregulation in immune responses over a course of 7 D MG infection in chickens. Various inflammation and immune-related signaling pathways including TLR, mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways were differentially expressed, depicting the complex inflammatory response during MG infection (Beaudet et al., 2017). Some other in vivo and in vitro studies examined inflammatory response, induction of apoptosis, and triggering of autophagy following MG infection (Majumder and Silbart, 2016; Tian et al., 2016; Wu et al., 2019; Bao et al., 2020; Zhang et al., 2020). The interaction and modulation of host immune responses is of major importance in the preparation of effective vaccine against MG infection. More importantly, infection by other respiratory pathogens must be taken into account in preparation of vaccine against MG. For instance, several studies reported co-infection of MG with influenza virus, Escherichia coli and infectious bronchitis virus (Stipkovits et al., 2012; Sid et al., 2016; Bwala et al., 2018; Awad et al., 2019; Canter et al., 2019; Wu et al., 2019). A previous report assessed the efficacy of MG vaccine in a co-infection model and suggested that ts-11 and 6/85 provided protection up to some extent against virulent MG strain. The 2 vaccines provided nonspecific protection. Whereas, the ts-11 proved to be more effective than 6/85 vaccine in trachea, bursa, and air sacs but not in lungs (Bwala et al., 2018). In addition, a recombinant adenovirus-based vaccine candidate was developed and evaluated for co-infection against infectious bronchitis virus and MG in chickens. A recombinant adenovirus was constructed containing the TM-1 protein of MG (pBH-S1-TM-1-EGFP) and S1 spike glycoprotein of infectious bronchitis virus. The recombinant bivalent pBH-S1-TM-1-EGFP adenovirus construct vaccine provides potential protective effects against bronchitis and MG infection (Zhang et al., 2018). Therefore, preventive measures against MG infection and other respiratory pathogens must take into account by vaccination and/or antimicrobial agents. The use of a challenge model in vaccination trial that reproduces the disease also plays a crucial role. There are several other factors and challenges that affect the potency of vaccine such as strain of MG, dose, route, and age of animals that are necessary to be considered during developing a vaccine against MG infection (Levisohn and Kleven, 2000; Branton et al., 2002; Evan et al., 2005; Evans et al., 2009). These challenges are often ignored in the preparation of vaccine, and therefore, care must be taken to overcome these challenges to successfully prevent and eradicate this devastating organism.

MG vaccine candidates

Till now, the vaccines available for MG infection consist of live-attenuated vaccines, inactivated vaccines such as oil-emulsion bacterins. Some of the commonly used vaccines available in the world are listed in Table 1. Adler for the first-time suggested vaccination against mycoplasmas infections in poultry in 1960 (Adler et al., 1960). Later on, Luginbuhl et al. and Fabricant used it in field trials (Luginbuhl et al., 1967; Fabricant, 1975). A large number of studies focused on vaccination against MG infection, which is mostly used in commercial flocks, particularly in broiler breeder and layers (Whithear, 1996). A study proved that immunization with vaccine partially depended on bursal-dependent lymphoid cells but not on thymus (Lam and Lin, 1984). Recently, a study reported the possible differences in host response between F-strain–based vaccines. The genomes of the F99 parent strains and AviPro vaccine were sequenced for comparison with the already sequenced F-strain vaccine (Leigh et al., 2019). Control of MG infection needs investigation of MG-infection free flocks. For this reason, rapid and cost-effective PCR-based assays were developed for the simultaneous detection of ts-11, 6/85, and F vaccine strains from field isolates (Sulyok et al., 2019). There are 2 main candidates of MG vaccine known as live-attenuated vaccine and killed vaccine/bacterins.

Live-Attenuated Vaccines

Three live MG vaccines were commercially approved including 6/85 strain, ts-11, and F strain. The 3 strains have effectively reduced losses related with MG infection in the field (Carpenter et al., 1981; Whithear et al., 1990a, 1990b; Evans and Hafez, 1992) and different in protection afforded, pathogenicity, and transmissibility (Branton et al., 2002). The F-strain vaccine was found to be efficacious and virtually nonpathogenic under field conditions (Levisohn and Kleven, 1981). Another study reported that adjusting for strain differences, the average egg production of uninfected flocks, MG (−), was 8.7 eggs hen housed greater than that of MG (vacc) flocks; production was 222.9 and 214.2, respectively (Carpenter et al., 1981). In addition, F strain vaccination reduced antibiotic requirements and mortality (Luginbuhl et al., 1967; Branton and Deaton, 1985; Self, 2003). F strain is preferable on sites where wild-type MG is very virulent and has the potential to displace a virulent MG strain in a commercial flock (Kleven et al., 1990). However, the disadvantages associated with the F strain is pathogenic and transmissible to broilers and turkeys (Vance et al., 2008). The other MG vaccines, ts-11 and 6/85, were found safer because of less pathogenicity and transmissibility toward young broilers and turkeys. While, these strains were found less effective in field challenge than F strain (Abd-El-Motelib and Kleven, 1993; US Animal Health Association, 2002). The effects of 6/85 or ts-11 strain vaccine on production have not been extensively investigated. It has been demonstrated that a single vaccination is often enough for a layer flock. Both 6/85 and ts-11 are preferable to F strain vaccine because of their low potential spread and risk to nearby unvaccinated flocks and safety characteristics. However, secondary vaccinations have been done in previously vaccinated flocks. For instance, a layer flock vaccinated with ts-11 or 6/85 have been revaccinated because of MG breaks with F strain (Gingerich, 2002). The F strain vaccine can be administered as early as 2 wk before infection by intranasal and intraocular and by coarse spray (Levisohn and Kleven, 2000). Another novel live-attenuated MG vaccine (K5054) was isolated from turkeys and proved to be effective in turkeys and chickens against virulent strains of MG (Ferguson et al., 2003). Further research can investigate this strain as an effective and safe vaccine for both chickens and turkeys. However, a limiting factor is the ability of these strains used in vaccines to revert to their pathogenic counterparts. Recently, vectors or carriers have gained immense importance in protecting against pathogens that are localized in the mucosal respiratory tract of the host (Mingozzi and High, 2013; Athanasopoulos et al., 2017; Humphreys and Sebastian, 2018). Bacteria such as both gram-negative and gram-positive have been used as carriers of recombinant antigens. Nonpathogenic and attenuated bacteria may be used as vectors, but nonpathogenic species are preferred (Liljeqvist and Stahl, 1999). The development of recombinant vaccines by cloning and identification of important MG surface antigens and colonization factor, development of expression and transformation strategies bewitch the interest of scientist (Zhang et al., 2010, 2018; Shil et al., 2011). Gene transfer is accomplished in mycoplasma species by electroporation (Liu et al., 2000; Pour-El et al., 2002; Mudahi-Orenstein et al., 2003) and Enterococcus faecalis–mediated conjugation (Ruffin et al., 2002). The transposons Tn4001 and Tn916 were found functional in mycoplasma (Knudtson and Minion, 1993; Cao et al., 1994; Dybvig et al., 2000; Pour-El et al., 2002; Mudahi-Orenstein et al., 2003) and can be used in mutant construction, protein functional analysis, cellular tagging, and gene expression (Dybvig and Voelker, 1996). For example, wild-type GapA, multiple genes encoding surface antigens and putative colonization factors have been characterized and cloned, and LacZ fusion protein studies were completed to date (Hnatow et al., 1998; Boguslavsky et al., 2000; Papazisi et al., 2002a; Liu et al., 2002; Jenkins et al., 2007; Yavlovich et al., 2007; Qi et al., 2018). The use of these proteins and technologies in recombinant vaccine can provide an effective and safe MG vaccine, but it would be complicated because of variable expression of these proteins. Recently, an attenuated and genetically modified MG strain known as GT5 was developed in laboratory (Mohammed et al., 2007; Gates et al., 2008). An in vivo study investigated that GT5 vaccinated chickens were from MG-induced disease. Specifically, the infection-associated lesions in trachea and colonization abilities of virulent MG strain were reduced (Papazisi et al., 2002b). Different vehicles serve for expression of MG antigens and recognized by host immune system. To this end, live-attenuated viruses also used as vehicles. For instance, USDA-approved recombinant fowl pox virus encoding genes for MG and used in turkeys and chickens. Studies demonstrated that chickens vaccinated with the vaccine containing vehicle fowl fox virus produced desirable effects and protected chickens from MG challenge (Evan et al., 2005). However, further investigation is necessary to determine the level of protection by vaccine in which virus is used as a vehicle. Another recombinant vaccine is the expression of MG antigens in nonpathogenic bacteria, with the benefits of vector effectivity, stability and safety, and selection of a proper host. The limitation is that the nonpathogenic strains have poorly characterized genetic systems that are residing in the avian respiratory tract. For example, MG was considered as nonpathogenic, isolated from the respiratory and reproductive tract of chickens (Wang et al., 1990), and evaluated as a possible vector for MG antigen expression (Evan et al., 2005). Recently, the efficacy of K-strain as a live-attenuated vaccine was reported to be safe in chickens (Ferguson-Noel and Williams, 2015). Moreover, another study compared the protection elicited by K-strain and ts-11 in layers and efficacy of K-strain and F-strain in broilers. Their results showed that K-strain vaccine has equivalent efficacy and potential to protect vaccinated birds from field challenge (Ferguson-Noel and Williams, 2015). F strain vaccine was administered in ovo to layers to reduce cost of labor and evaluate it as an effective vaccination to control MG infection in commercial layer flocks. However, the results showed higher embryo mortality rate and further research is needed to investigate and optimize the dose and immune response offered by this method (Elliott et al., 2017). The short-term and long-term efficacies of MG vaccines, strains ts-11 and 6/85, were demonstrated in chickens through aerosol and eye drop inoculations. The results showed the level of protection offered by the 2 vaccines were found similar after 36 wk postvaccination (Noormohammadi and Whithear, 2019). A live-attenuated vaccine named Vaxsafe MG (strain ts-11) has been reported to provide protection in chickens. The study proved that the vaccine can also be used in turkeys and found safe and efficacious in 3-wk-old turkeys (Kanci et al., 2018). While, limited information is available about the use of Mycoplasma gallinarum as a vector and host, and therefore, further studies are encouraged to scrutinize the organism's genetic system in detail.

Killed/Inactivated Vaccines/Bacterins

More complex vaccines consist of 1 or more purified antigens, killed pathogens, or bacterins with an oil adjuvant that stimulate the immune response. These vaccines are protective, but their use is limited because of cost (Ley, 2003). Bacterins are inactivated so these vaccines are safer than live vaccines. Studies demonstrated that bacterins were efficacious in prevention of respiratory lesions in chickens and proved beneficial in reducing transmission and production losses (Rimler et al., 1978; Yoder, 1979, 1983; Hildebrand, 1985). However, some researchers have demonstrated that bacterins were not so effective as live vaccines, as bacterins can temporarily control MG infection and have negligible effect in protecting host respiratory system from MG (Kleven, 1985). Therefore, bacterins are of minimal value in commercial flocks where long-term control of MG infection is needed. In addition, bacterins are costly and expensive and difficult to vaccinate as each bird need individual vaccination, and often 2 doses are needed as repeated vaccination found more effective than single dose.

Conclusions and future directions

The use of vaccines is an alternative means for the prevention and control of MG, particularly in commercial layer and breeder flocks with the benefits of avoiding considerable economic losses. Live-attenuated vaccines such as F strain, ts-11 strain, and 6/85 strain and inactivated vaccines such as bacterins are commonly used in commercial flocks, but the sudden onset of MG infection could occur in some circumstances. For instance, latent infections and transmission both by horizontal and vertical means are possible among different and same flocks. Therefore, biosurveillance and biosecurity practices are recommended to carry out for the complete eradication of infected flocks. Live vaccines often showed pathogenicity and adverse side-effects, whereas bacterins have high cost, and often repeated doses are required to boost avian immune system. Hence, new novel recombinant vaccines are needed to be developed which are more efficacious and less expensive. Limited information is available about the type of immune response, key protective antigens, and optimal adjuvant formulations. Research on all possible antigens for their potential through reverse vaccinology (Rappuoli, 2000) may be needed as this is more advanced method to identify recombinant proteins that provide protection against experimental challenge (Perez-Casal et al., 2015; Nkando et al., 2016). Research demonstrated that MG vaccination in turkeys and chickens produced a high antibody titer including increased CD4+ and CD8+ expressing lymphocytes in trachea. This suggested that these cells contributing toward the pathogenesis of the disease (Wijesurendra et al., 2017). Therefore, agents which enhance the production of humoral immune response including lymphocytes production could be considered for the possible protection trial of MG infection in future studies. In addition, research on in silico approaches had not yet been reported for MG. While, some researchers used in silico approaches for the identification of virulence candidates for other mycoplasma species such as Mycoplasma pneumoniae type 2a strain 309 and Mycoplasma agalactiae (Shahbaaz et al., 2015; Gaurivaud et al., 2016). Thus, future studies are recommended on in silico approaches for the development of effective vaccines. Besides, several studies focused on adjuvants such as chitosan and other adjuvants to boost host immune responses infected with MG (Barbour and Newman, 1990; Limsatanun et al., 2018). The role of adjuvants in enhancing immune responses cannot be neglected. Adjuvants increased the immunogenic responses of antigens and enhanced the efficacy and potency of vaccines (Bastola et al., 2017). Recently, studies reviewed the fundamental knowledge for the application of adjuvants in vaccines (Kwissa et al., 2007; Riese et al., 2013; Jin et al., 2019). However, limited information is available about adjuvants used for MG vaccine preparation (Barbour and Newman, 1989, 1990; Hussein et al., 2007; Limsatanun et al., 2018). Hence, it is of prime importance to screen new and more effective adjuvants for potential benefits and effective vaccine preparation to control MG infection. Moreover, a strong support is needed from funding organizations to invest more in research associated with vaccine preparation against MG infections, as these researches are necessary for the elucidation of molecular mechanisms required for the development of effective vaccines or new treatments to protect from MG infection.