Introduction
Hemorrhagic septicemia is a short course, lethal and
septicemic disease of buffaloes and cattle caused by a Gram negative,
non-motile, coccobacillus bacterium, the Pasteurella
multocida (Boyce et al. 2000). HS is a main epizootic disease in
cattle and buffaloes in several countries with high morbidity and mortality
(El-Jakee et al. 2016). In Asia, HS has caused severe economic losses
(Benkirane and De Alwis 2002; Abba et al. 2017).
Treatment of HS with antibiotics is prolonged, expensive
and ineffective because of emerging resistance of antibiotics against P. multocida. The use of antibiotics
might cause toxicity to human consumers. Acute nature of HS is another
hindrance in treatment (Ahmad et al.
2014). Different vaccine types including oil adjuvanted, multiple
emulsion and alum precipitated vaccines are used to control this fatal disease.
Killed vaccines have been extensively used against HS in the world
(Shivachandra et al. 2011). Nonetheless, these injectable vaccines are
difficult to administer as restraining the animals has been found to be very
difficult (Sarah 2007). Moreover, oil adjuvant vaccine is fairly disliked by
farmers because it possesses dense viscosity (Sarah et al. 2006; De
Alwis 1992). The application of live vaccine that can be given intra-nasally is
a substitute method to save the animals from HS.
Live vaccines have played a pivotal part from the start
of immunology (Detmer and Glenting 2006). Yet, there has been failure to
develop vaccine having greater viable inhabitants of live microorganism for
protection from disease. Thus, the initiation of an effective and systematic
formulation approach is critical to increase survival rate, storage stability
and bacterial cell activity in live vaccine. Freeze-drying or lyophilization is
a convenient technique to conserve bacteria and viruses by dehydrating cellular
fluid (Das et al. 2018). The purpose of lyophilization is to convert
bacteria in a stable form which can be stored for a longer period of time.
Moreover, it is mainly required to attain high viability of cells (Winters and
Winn 2010). At present, freeze drying is a frequently used method for this
purpose. It has been reported that many strains of bacteria have shown better
survival rates after lyophilization for long storage duration (Peiren et al.
2015). This biophysical process is relatively comfortable and generally used
for cultural collection of microorganisms. The discovery also narrates the way
for preparing a stabilized product, vaccine (Morgan et al. 2006).
However, this method might render the microorganisms to extra stressful
operational steps therefore dropping their viability (Saarela et al.
2005; Schoug et al. 2006). The procedure principally involves freezing,
sublimation and lastly desorption of the water molecules from the
microorganisms (Stephan et al. 2016). Process of Freeze drying contains
three basic steps: first is the freezing, second is primary drying and third is
secondary drying. During the first step, the freezing, water is transformed
into ice, generally entrapped in the amorphous medium of the lyoprotectant, a
material that prevents cells from damage throughout this process. The freezing
temperature of amorphous lyoprotectant must be below its glass transition
temperature (Tg’) to make sure an entirely solid product. During the primary
drying, ice is eliminated through sublimation. When the primary drying
temperature of the product is excessively high (i.e., above the collapse
temperature Tc), the porous product can disintegrate, therefore high
temperature should be avoided (Fonseca et al. 2004). During secondary
drying, remaining unfrozen water captured in the glassy medium is eliminated by
isothermal desorption. The endpoint of the secondary drying step is set to get
preferred residual moisture content (RMC). Death or inactivation of
freeze-dried bacterial cells during storage is reliant on water activity (aw),
storage temperature and residual moisture content (Aschenbrenner et al.
2012; Passot et al. 2012).
Protectant gives steadiness and safety against further
inactivating processes such as protein denaturation which is relatively
sensitive and reduces viability of several cell types (Carvalho et al.
2004). For the ease of use and to ensure that the vaccine product is in its
optimum functional and efficient form, various sugars for instance lactose,
sucrose, glucose and trehalose have been frequently used as protective means
(Hubalek 2003). Apart from, skimmed milk and nitrogen compounds such as
peptone, yeast extract and casein hydrolysate have also been studied (Berny and
Hennebert 1991). These protective agents perpetually play a pivotal part in
consenting cells to be managed for storage at deep temperatures and to be
restored with suitable viability (Elliott et al. 2017).
In this study, three different stabilizers: trehalose,
lactalbumin and skimmed milk were used to evaluate viability of P. multocida B:3,4 strain during freeze
drying process. Furthermore, combination of these stabilizers in suitable
concentrations was also used for this purpose. The conservation of cell
viability is first and foremost requirement to produce a live vaccine because
vaccination is an effective and economical practice to prevent infectious
diseases. The present study was conducted to evaluate the effect of three
stabilizers to conserve the viability of P.
multocida B:3,4 during lyophilization and subsequent storage at four
different temperatures. This stability study of P. multocida B:3,4 will
play a key role for the determination of suitable stabilizer and storage
temperature that ensure the safety, efficacy and viability of live HS vaccine.
Materials
and Methods
Source
of the strain
The strain of P. multocida B:3,4 was retrieved
from the inventory at -20°C maintained at Bacteriology laboratory of Animal
Health Program, Animal Sciences Institute, National
Agricultural Research Center (NARC), Islamabad.
Revival
and growth of microorganisms
Mice inoculation method was used to revive P.
multocida B:3,4. The method was adopted according to Singh et al. (2010) with minor modifications. Swiss
albino mice (n=5) were purchased from Animal House, National Institute of
Health (NIH), Islamabad. The mice were kept and reared in lab animal house,
Animal Health Program, NARC, Islamabad. These mice were divided into two
groups, Group-I (n=2) and Group-II (n=3). Lyophilized P. multocida B:
3,4 strain was reconstituted in 1 mL normal saline (pH=7.2). Reconstituted
strain was streaked on tryptic soya agar (TSA) and incubated at 37°C for 18 h.
After incubation Group-I was injected intraperitoneal with 200 µL of 10-5
dilution P. multocida B:3,4 culture. Group-II was injected with 200 µL of
normal saline solution. After 24 h, inoculated mice were found dead. However,
mice of control group remained alive. The dead mice were dissected and spleen
and heart were collected aseptically. Morbid organs (heart and spleen) were
used to streak on blood agar. Heart blood was also collected and used for streaking on blood
agar. These streaked plates were incubated for 24 h at 37°C. After
incubation, the resultant colonies were identified by Gram’s staining. The
isolated colonies were selected and harvested on blood agar. The plates were
incubated at 37ºC for 24 h to get pure cultures. Different biochemical tests,
differential media i.e; MacConkey agar and PCR were used (Zhao et al. 2019) for confirmation of P. multocida strain B: 3,4.
Preparation
of stabilizer media
Three stabilizers, trehalose (Sigma-Aldrich Co),
lactalbumin (Neogen Corporation, Michigan) and skimmed milk (Neogen Corporation,
Michigan) were selected (Gehrke et al. 1992; Conrad et al. 2000;
Chen and Kristensen 2009) to check their effect on viability of P. multocida
B:3,4. They were evaluated using 5, 10, 15 and 20% concentrations (Oslan et
al. 2017; Bassiouny et al. 2019). To prepare stabilizer media,
desired concentrations of stabilizers namely skimmed milk, lactalbumin and
trehalose were suspended in distilled water. Skimmed milk and lactalbumin were
autoclaved at 121°C for 15 min before mixing with the P. multocida B:3,4. Trehalose was sterilized by filtration using
microfilter having pore size 0.20 µm. A control was prepared by suspending P. multocida B:3,4 with autoclaved
distilled water.
Preparation
of bacterial suspension
Bacterial suspension was prepared by inoculating pure
culture of P. multocida B:3,4 in tryptic soya broth (TSB). Suspension
was vortexed for few seconds to make it homogenous. Afterward, suspension was
incubated at 37ºC for half an hour.
Viability
before lyophilization
Equal volumes (1:1) of desired concentrations (Bora et al. 2015) of stabilizers and P. multocida B: 3,4 enriched in TSB were
mixed in 5 mL freeze drying vials (1 mL filled volume). The viability of
samples before freeze drying was determined by Miles and Misra method (Miles et al. 1938) and expressed as
colony forming units (CFUs) per mL. Suspensions were frozen in glass vials by keeping
at -80ºC for at least one hour before lyophilization.
Lyophilization
Process of lyophilization (freeze drying) was completed
in 48 h cycle. Suspension of P. multocida
B: 3,4 was cooled in a shelf at a linear ramp rate in pilot scale lyophilizer
(Ilshin BioBase, Europe). A conventional freeze-drying process completes in
three steps; freezing, primary drying and secondary drying (Tang and Pikal
2004). After completion of lyophilization, the viability of lyophilized samples
of P. multocida B: 3,4 was determined using Miles and Misra Method (Miles et al. 1938) and stated as CFU/mL.
Storage
at different temperatures
Lyophilized vaccine was stored
at different temperatures i.e., -20, 4, 25 and 37°C to check the effect of temperature on viability of P.
multocida in various stabilizers (Oslan et al. 2017).
Revival,
enumeration and determination of cell viability
The viability of cells after lyophilization was
calculated as CFU. Three vials of each concentration were taken to determine
viability i.e. readings were taken in
triplicate to calculate mean. Before determining CFUs, freeze dried samples
were reconstituted by adding 1 mL phosphate buffer saline (PBS) adjusted at pH
7.2. To get homogenous solution, the samples were vortexed and then incubated
at 37°C for 30 min to make sure the absolute dissolution of bacterial material
and PBS. Miles and Misra method (Miles et al.
1938) was adopted to determine CFUs. Briefly, lyophilized vaccine was
reconstituted in 1 mL PBS (pH 7.2). Then vaccine was vortexed for few seconds
to obtain homogenous suspension. The reconstituted vaccine (1 mL) was added to
9 mL of PBS to make serial dilutions up to 10-10. From each serial
dilution, 20μl was inoculated on TSA in triplicate. The plates were kept
undisturbed until inoculated suspension was completely immersed by the agar.
Plates were then incubated for 24 h at 37ºC. After incubation, the number of
colonies was calculated. The percentage of viable cells was estimated using
following equation:
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Results
Effect of
duration of lyophilization on viability of P. multocida B:3,4
Initially, lyophilization was conducted for 24 h. After
24 h, vials were removed from lyophilized and observed. Lyophilization involves
conversion of microorganisms from a liquid state to a solid compact mass
however using 24 h duration partial lyophilization with moisture contents was
found. It indicated that 24 h duration was not able to convert the bacterial
suspension into a compact mass. Hence, one more freeze-drying cycle of total 48
h was tried aimed to attain homogenous freeze-dried mass. The vials after 48 h freeze
drying showed compact mass with no moisture contents which indicated the
complete lyophilization. Results of viability of both cycles are explained in
Table 1. First cycle of freeze drying using 24 h duration was found unable to
protect the viability of P. multocida B:3,4 and caused substantial loss
of viability. The survival rates were reduced to 0%. The survival rate of cells
was significantly higher using 48 h duration with different stabilizers.
Results indicated that amongst protective agents, trehalose, skimmed milk and
lactalbumin at the concentration of 5% the percentage of viability was 32.39%±1.9,
0.036%±0.002 and 26.08%±0.81 while at the 10% concentration survival rate was
47.22%±1.19, 0.58%±0.02 and 31.42%±0.62 percent respectively (Table 1). The 15%
concentration showed survival rates for trehalose, skimmed milk and lactalbumin
corresponding to percentage viability of 89.87%±1.53, 0.81%±0.020 and 81.81%±1.42
respectively. On the other hand, the 20% concentration of trehalose, skimmed
milk and lactalbumin showed percentage of viability 82.89%±2.3, 0.82%±0.024 and
76.92%±2.02 respectively. The stabilizers demonstrated maximum viability after
freeze drying at the concentration of 15% using 48 h duration of
lyophilization. For this reason, 15% concentration of all stabilizers was used
in succeeding investigation.
Effect of
stabilizers on viability of P. multocida B:3,4
Table 1: Effect of
freeze-drying cycle on the viability of P. multocida B:3,4 using three
different stabilizers i.e., trehalose, lactalbumin and skimmed milk
|
Duration of freeze-drying cycle |
Protective agent |
Percentage used |
Before freeze drying (CFU/ml) |
After freeze drying (CFU/ml) |
% of viability ± S.D |
|
24 h |
Distilled water |
Control |
7.3×1010 |
4.2×104 |
0.000057±2.1×10-6 |
|
Trehalose |
5% |
7.4×1010 |
4.4×105 |
0.00059±1.4×10-5 |
|
|
10% |
6.7×1010 |
8.4×104 |
0.00012±0 |
||
|
15% |
7.1×1010 |
8.0×106 |
0.011±0.0008 |
||
|
20% |
6.5×1010 |
7.6×104 |
0.00011±4.7×10-6 |
||
|
Skimmed Milk |
5% |
6.4×1010 |
4.1×104 |
0.000064±3.09×10-6 |
|
|
10% |
7.2×1010 |
4.4×104 |
0.000061±1.4×10-6 |
||
|
15% |
7.8×1010 |
6.2×105 |
0.00079±1.6×10-5 |
||
|
20% |
7.9×1010 |
5.1×104 |
0.000064±4.7×10-7 |
||
|
Lactalbumin |
5% |
7.2×1010 |
2.1×104 |
0.0000072±3×10-5 |
|
|
10% |
7.9×1010 |
4.3×104 |
0.000054±2.4×10-6 |
||
|
15% |
7.4×1010 |
6.3×105 |
0.00085±1.2×10-5 |
||
|
20% |
6.1×1010 |
3.7×104 |
0.000060±1.6×10-6 |
||
|
48 h |
Distilled water |
Control |
7.4×1010 |
4.6×104 |
0.000062±2.8×10-6 |
|
Trehalose |
5% |
7.1×1010 |
2.3×1010 |
32.39±1.9 |
|
|
10% |
7.2×1010 |
3.4×1010 |
47.22±1.19 |
||
|
15% |
7.8×1010 |
7.1×1010 |
89.87±1.53 |
||
|
20% |
7.6×1010 |
6.3×1010 |
82.89±2.3 |
||
|
Skimmed Milk |
5% |
6.6×1010 |
2.3×107 |
0.036±0.002 |
|
|
10% |
7.3×1010 |
4.3×108 |
0.58±0.02 |
||
|
15% |
7.6×1010 |
6.2×108 |
0.81±0.020 |
||
|
20% |
7.5×1010 |
6.2×108 |
0.82±0.024 |
||
|
Lactalbumin |
5% |
6.9×1010 |
1.8×1010 |
26.08±0.81 |
|
|
10% |
7.0×1010 |
2.2×1010 |
31.42±0.62 |
||
|
15% |
7.7×1010 |
6.3×1010 |
81.81±1.42 |
||
|
20% |
7.8×1010 |
6.0×1010 |
76.92±2.02 |
Table 2:
The viability of P. multocida B:3,4 cells formulated with 15 % (w/v) of
protective agents (trehalose, lactalbumin and skimmed milk) after freeze drying
|
Protective agent (15% w/v) |
Viable cells CFU/mL |
Percentage of viability ± S. D |
|
|
Before freeze drying |
After freeze drying |
|
|
|
Distilled water (control) |
7.8×1010 |
3.2×106 |
0.004±0 |
|
Trehalose |
7.4×1010 |
6.8×1010 |
91.89±0.08 |
|
Skimmed Milk |
7.2×1010 |
3.5×108 |
0.47±0.009 |
|
Lactalbumin |
7.6×1010 |
6.1×1010 |
80.38±2.57 |
|
Trehalose (15%) + skimmed milk (15%) |
7.5×1010 |
3.2×1010 |
43.1±0.86 |
Table
3: Viability of freeze dried P. multocida B:3, 4
cells using three different protective agents (trehalose, lactalbumin and
skimmed milk) during storage at -20⁰C, 4⁰C, 25⁰C and 37⁰C for 30 days
|
Protective agent (15% w/v) |
Day 0 |
Day 30 |
||||||||||
|
|
-20⁰C |
4⁰C |
25⁰C |
37⁰C |
-20⁰C |
% viability ±S.D |
4⁰C |
% viability ±S.D |
25⁰C |
% viability ±S.D |
37⁰C |
% viability ±S.D |
|
Distilled water |
3.4×107 |
3.5×107 |
3.3×107 |
3.5×107 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Trehalose |
6.2×1010 |
6.6×1010 |
6.3×1010 |
6.3×1010 |
5.4×1010 |
84.91±2.5 |
3.4×1010
|
52.50±2.79 |
6.1×109 |
9.74±0.41 |
5.3×108 |
0.84±0.04 |
|
Skimmed Milk |
6.6×1010 |
6.3×1010 |
6.4×1010 |
6.7×1010 |
3.7×108 |
0.55±0.009 |
2.6×105 |
0.00041±2.05×10-5 |
0 |
0 |
0 |
0 |
|
Lactalbumin |
6.1×1010 |
6.2×1010 |
6.4×1010 |
6.1×1010 |
4.5×1010 |
73.36±2.03 |
2.3×1010 |
37.81±3.7 |
3.3×109 |
5.22±0.16 |
0 |
0 |
|
Trehalose+ skimmed milk |
6.7×1010 |
6.4×1010 |
6.5×1010 |
6.3×1010 |
2.6×1010 |
38.87±4.13 |
6.2×109 |
9.68±0.33 |
5.2×106 |
0.0079±4.7×10-5 |
0 |
0 |
The survival rate of P. multocida B:3,4 with
three different stabilizers at 15% concentrations after lyophilization is shown
in Table 2. The highest percentage (91.89%±0.08) of viable cells was attained
with 15% (w/v) trehalose as the stabilizer. Lactalbumin (15%, w/v) showed an
agreeable percentage of viability as 80.38%±2.57. Skimmed milk as stabilizer
did not prevent the viable cells from damaging effects of freeze-drying
process. There was an enormous difference of viable cells of P. multocida B:3,4
after lyophilization using skimmed milk as stabilizer. The viability in skimmed
milk was 0.47%±0.009 after freeze drying. Additionally, skimmed milk (15%) was
mixed with trehalose (15%) to check the combined effect of both stabilizers on
viability. The percentage of CFUs was markedly improved (43.1%±0.86) as
compared to skimmed milk when it was used alone as cryoprotectant (0.47%±0.009).
However, cell viability of lyophilized cells using combination of both
stabilizers was still too inferior to use as protective agent. The findings of
this experiment proposed that skimmed milk alone or its combination with
trehalose is not appropriate for the conservation of viability of P.
multocida B:3,4 during lyophilization procedure. However, trehalose was the
only protectant that showed highest survival rate in this study. Therefore,
from these results, it can be demonstrated that trehalose, a disaccharide, is
effective to be used as stabilizer during freeze drying of P. multocida
B:3,4.
Effect of
storage temperature on survival rate of lyophilized P. multocida B:3,4
After rehydration in PBS, freeze dried P. multocida
B:3,4 in three stabilizers was stored at four different temperatures -20, 4, 25 and
37°C to check the survival rate. Three vials for each temperature were
stored to take readings in triplicate. The viability of P. multocida
B:3,4 before freeze drying in the protective agents at day zero and day 30
after storage at -20, 4, 25 and 37°C was checked. The results (Table 3) showed that
84.91±2.5 of cells remained viable at -20⁰C using trehalose 15% (w/v) as
stabilizer after one-month storage. The 4°C storage temperature showed 52.50%±2.79 of survival rate with same protective agent. Whereas
other two storage temperatures 25 and 37°C showed viability of 9.74%±0.41 and 0.84%±0.04 respectively. Lactalbumin
(15%, w/v) showed viability of 73.36%±2.03 at -20°C storage temperature while at 4°C survival
rate was 37.81%±3.7. The viability was found very low (5.22%±0.16) at 25°C and at 37°C it dropped
to 0% in lactalbumin (15%). The results showed that skimmed milk (15%, w/v) was
unable to protect the viable cell at all four storage temperatures. After
one-month storage at -20, 4, 25 and 37°C in skimmed milk, the viability declined to 0%. When a
combination of skimmed milk (15%) and trehalose (15%) was used, comparatively
an enhanced survival rate was obtained (38.87%±4.13) at -20°C as compared
to viability of individual skimmed milk (0.55%±0.009). However, this combination
showed very low survival rates at 4⁰C (9.68%±0.33) while results at 25 and 37°C revealed
that viability has been declined to 0%. These results demonstrated that
different storage temperatures significantly affected the viability of P.
multocida B:3,4 in various protective agents. It was obvious that among all
these storage temperatures, -20°C is most suitable storage temperature to conserve
viability following 4°C temperature. Whereas, 25 and 37°C have
detrimental effects on survival rate of P. multocida B:3,4. Among three
stabilizers, trehalose was found the most appropriate stabilizer that achieved
highest survival rate of P. multocida B:3,4 after 30 days storage at -20°C and showed
the least viability reduction from 6.4×1010 to 5.4×1010
CFU/mL. Our results showed that trehalose provided protective effect on
lyophilized P. multocida B:3,4 at -20°C. Therefore,
trehalose is the best stabilizer to conserve viability of P. multocida B:3,4 and -20⁰C is most suitable storage
temperature of for this microorganism. Consequently, trehalose was selected as
stabilizer to prepare live aerosol HS vaccine and storage temperature
-20⁰C was selected for preservation.
Discussion
To our knowledge, this is first study on effect of
stabilizers and storage temperature explaining viability of freeze-dried P. multocida B:3,4.
Use of bacterial cells as live vaccine for disease prevention depends on the
conservation and preservation of viable cells which are required to ensure long
term delivery of stable vaccine in terms of viability (Jain et al.
2020). Therefore, a live vaccine targets include specific number of CFUs,
conservation of live cells during lyophilization, and retaining the viability
during production procedure and storage (Saarela et al. 2000; Lacroix
and Yildirim 2007; Mahapatra et al. 2020). The delivery, storing and use
of vaccines consequently present challenges that could be reduced by enhanced
stability achieved by lyophilization and adding stabilizers with resulting
betterment in vaccine efficiency. The aim of this work was to assess the
protective effect and suitable concentrations of trehalose, skimmed milk and
lactalbumin during lyophilization and storage of P. multocida B:3,4 to
develop live HS vaccine. The selection
of protectant for specific organism that might preserve viability during
lyophilization and storage is essential. In this study trehalose, skimmed milk
and lactalbumin were selected because these most commonly used
stabilizers are able to protect viability of the vaccines during lyophilization
and even during storage after rehydration with diluent (Gehrke et al.
1992; Conrad et al. 2000; Chen and Kristensen 2009; Bellali et al.
2020). Moreover, these three stabilizers are widely used to conserve number of
organisms (Zhang and Hui 2017; Latif et al. 2018). The studies on
stability of microorganisms have shown that these three stabilizers contain
protective effects on the viability of microorganisms (Mariner et al.
2017; Bora et al. 2018). Therefore, these stabilizers were selected on
the basis of previous studies on several other bacteria.
The results from duration of lyophilization showed that 48 h cycle of
freeze drying was the most appropriate approach as compared to 24 h duration.
The percentage of viable cells was significantly greater in cycle of 48 h duration.
The primary drying step in freeze drying is conversion of liquid culture into
ice-crystals and then to remove ice in the form of vapors (Nireesha et al.
2013; Pansare and Patel 2019). In cycle 1 (24 h), time for removal of ice
crystals during lyophilization was shorter than cycle 2 (48 h) which was resulted in the development of ice residuals that appeared in the
form of moisture in P. multocida
B:3,4 during 24 h duration (Saclier et
al. 2010). A previous study conducted by Oslan et al. (2017) to
evaluate the effect of different stabilizers on the viability of mutant P.
multocida B:2 cells after lyophilization process using two different time
durations of cycles also indicated that freeze drying duration of 54 h was the
most appropriate approach in preserving cell viability of mutant P.
multocida B:2 compared to cycle of 24 h duration. Consequently, a
sufficient time period for freeze drying process is important to ensure
complete lyophilization and conservation of viable cells to produce live
vaccine.
Stabilizers and their concentrations significantly affect the viability
of micro-organisms during freeze drying (Zhao and Zhang 2005; Shokri et al. 2019). The results of this study also
indicated that viability of P. multocida B:3,4 after freeze drying was
different with various stabilizers. The highest viability of P.
multocida B:3,4 was
observed when 15% trehalose was used as stabilizer. Previous studies also
reported high viability of Salmonella
enterica, Lactobacillus salivarius and Pseudoalteromonas
nigrifaciens after freeze drying when trehalose was used as
stabilizer compared to lactose, sucrose, sorbitol, lactalbumin, skimmed milk
and ascorbic acid (Zayed and Roos 2004; Kang
et al. 2010; Zhang et al. 2020). Trehalose is considered an excellent osmolyte
with remarkable stabilizing effects on cells and preserves the viability of
cells in freeze dried as well as in solution state (Kaushik and Bhat 2003).
Other earlier investigations have also directed that trehalose prolongs the
stability of several vaccines (Bora et al. 2015). When microorganisms
are exposed to stress, trehalose helps them in retaining cellular integrity.
This is thought to occur by prevention of denaturation of proteins by
trehalose, which would otherwise degrade under stress (Jain and Roy 2009). Other stabilizers possess low or lack this
ability. A potential fact for the significant defensive effect of trehalose to
viable cells’ plasma membrane and protein is by removing water in
plasma membrane removed during the freeze-drying procedure and prevent unfolding
and aggregating proteins by the formation of hydrogen bonds with polar groups
of proteins (Leslie et al. 1995; Crowe et al. 2001; Bellali et
al. 2020). It has been demonstrated that the trehalose would defend cell
proteins against denaturation (Guowei et al. 2019). More important,
trehalose penetrates the cells and reduces the damaging effects of osmosis
during water loss and prevents the development of ice crystals and resultant
breakdown of plasma membrane (Cota and
Alvim 2018). Concentration of the stabilizer considerably affects the viability
of micro-organisms during lyophilization. For example, lower concentration of stabilizer may not be able to form required hydrogen
bonds to provide protection against negative effects of freeze drying (Mensink et
al. 2017). Similarly, higher concentration of stabilizers may prove toxic
to cells thus lowering viability (Bhattacharya
2018). Therefore, optimization of stabilizer concentration for optimum cell
viability is essential.
The results
from investigations on the storage of P. multocida B: 3,4 with selected
stabilizers at four different temperatures revealed that the highest viability
was obtained at storage temperature of -20oC in 15%
trehalose after one-month storage. Similar to our findings Oslan et al.
(2017) also reported that the survival of P. multocida B: 2 was highest
at -30oC compared to 4oC and 27oC after 6-month
storage at three different storage temperatures. In our studies, trehalose at
the concentration of 15% retained highest cell viability at storage temperature
-20oC while lactalbumin (15%) at the
same temperature also showed reasonable viability. Though, skimmed milk failed
to conserve viability and reduced the viability to zero percent at all four
storage temperatures. Our results also agree with previous work of Bolla et
al. (2011). However, a combination of trehalose and skimmed milk improved
the cell viability after storage at -20⁰C. Similarly,
in previous studies, Malik et al. (1993); Kanmani et al. (2011);
Oslan et al. (2017) and Archacka et al. (2019) also used
combination of these two stabilizers and found the increased cell viability.
The protectants in combination may suppress each other or they may develop an
additive or synergistic effect (Guowei et al. 2019). In our case, it has
been observed that the effect of skimmed milk using such combination was
superior to individual but percentage of cell viability was too low to use as
protectant.
These results
indicated that the
storage temperature -20⁰C was the most pertinent temperature that
conserved highest viability. In this study, difference in viability of cells
exhibited that certain stabilizers are more effective than others to protect
the P. multocida B: 3,4. The effect of storage temperature on viability
is due to the fact that in response to different temperatures, breakdown of
membrane and other proteins in bacterial cells occurs that affects the
viability (Gur et al. 2011; Liu et al. 2019). Zeng et al. (2009) has also suggested
that loss of viability is consequence of temperature induced plasma membrane
damage. As a result, the irreversible damage to bacterial cell membrane leads
to unviable cells (Cota and Alvim 2018).
Conclusion
The viability of P. multocida
B: 3,4 during lyophilization and subsequent storage is dependent on the
selection of stabilizer. The selection of a suitable protective medium and
storage temperature is crucial to attain highest percentage of viability as it
is the main factor that affects the stability of the live vaccines. In our
studies, during freeze drying process and subsequent storage of P. multocida
B: 3,4, we found that trehalose was the most appropriate protectant that
greatly influenced the survival rate of P. multocida B:3,4. The highest
survival rates of P. multocida B: 3,4 were observed when cells were
lyophilized and stored at -20⁰C, which is the optimum
storage temperature for preservation of P. multocida B:3,4. The most
effective concentration of trehalose was 15% in this study. Consequently, for
storage of P. multocida B: 3,4 live aerosol HS vaccine, trehalose can be
added as protectant. Moreover, duration of freeze-drying cycle also
significantly affected the survival rate of cells and therefore it should be
optimized and confirmed to ensure that final vaccine contains required number
of live cells. These findings would help in development of live aerosol HS
vaccine. Development of live aerosol vaccine would contribute to prevent
animals from HS. However, still there is a need for evaluation and
standardization of a stabilizer that keeps the live aerosol vaccine stable and
viable at room temperature.
Acknowledgement
Authors thank Agricultural Linkages
Program, PARC (Project No: AS-142) for financial assistance for this study.
Author Contributions
Sajid Mahmood Sajid: Planning of study, standardisation and
execution of lab protocols, collection and analysis of samples from
experimental animals, analysis of data and write up of manuscript, Arfan
Yousaf: Planning of study, analysis of data and write up of manuscript, Hamid
Irshad: Planning and execution of study, analysis of data and write up of
manuscript, Muhammad Arif Zafar: Planning of study and analysis of data, Saif
ur Rehman: Planning of study and write up of manuscript.
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