Research Article

Preparation of an Inactivated Polyvalent Vaccine Against Common Bacterial Pathogens Causing Bovine Mastitis and Evaluation of its’ Immunizing and Therapeutic Potentials

Rafik Soliman1*, Rafik H. Sayed2, Hanan Mahmoud3, Heidy Abo El-Yazeed1, Marsell Saad1, Shaimaa Abdelall Elsaady2, Khalid Sh3

1Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Cairo University, Egypt; 2Central Laboratory for Evaluation of Veterinary Biologics (CLEVB), Agricultural Research Center (ARC), Ministry of Agriculture, Abbasia, Cairo, Egypt; 3Animal Health Research Institute (AHRI), Agricultural Research Center (ARC), Ministry of Agriculture, Giza, Egypt.

Received | August 19 2022; Accepted | September 20, 2022; Published | April 10, 2023

*Correspondence | Rafik Soliman, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Cairo University, Egypt; Email: rafiksoliman108@cu.edu.eg

Citation | Soliman R, Sayed RH, Mahmoud H, El-Yazeed HA, Saad M, Elsaady SA, Sh K (2023). Preparation of an inactivated polyvalent vaccine against common bacterial pathogens causing bovine mastitis and evaluation of its’ immunizing and therapeutic potentials. J. Anim. Health Prod. 11(2): 99-108.

DOI | http://dx.doi.org/10.17582/journal.jahp/2023/11.2.99.108

ISSN | 2308-2801

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Bovine mastitis is an economically important disease that affects the dairy farms and the national budget in two ways; direct cost due to per-acute forms of mastitis and loss of cows through premature culling and indirect costs due to reduced milk production and quality, expensive antibiotic therapy, and the serious side effects of the used antibiotics. Excessive use of antibiotics may lead to complete removal of all bacteria, the pathogenic and non-pathogenic, an effect, which has been reported to be associated with a marked increase in severity of acute mastitis (Khaitsa et al., 2000, Schlegelov à et al., 2002);

Mastitis can be caused by a wide range of organisms, including gram-negative and gram-positive bacteria, mycoplasmas, and algae. Many microbial species that are common causes of bovine mastitis, such as E. coli, Klebsiella pneumoniae, Str. agalactiae and S. aureus also occur as commensals or pathogens of humans, whereas other causative species, such as Str. uberis, Str. dysgalactiae subsp. Dysgalactiae or S. chromogenes, are almost exclusively found in animals (Zadoks, et al., 2011). Environmental streptococci are predominant etiological agents of both subclinical and clinical forms of mastitis (Ferguson et al., 2007; Olde Riekerink et al., 2007). These pathogens are now emerging as one of the most frequent causes of bovine mastitis (Ebrahimi et al., 2008).

The subclinical form of mastitis is one of the most persistent and widely spread disease conditions of importance to milk hygiene and quality among dairy cattle (Mørk et al., 2007; Ogola et al., 2007; Jaime Romero et al., 2018).

Since the introduction of control measures to decrease the prevalence of contagious mastitis pathogens like S. aureus and Str. agalactiae, little progress has been achieved. The environmental pathogens Str. uberis, Str. dysgalactiae, and Enterococcus spp. (fecal streptococci) are ubiquitous in farms and cause major management difficulties. Although environmental streptococci can be cultured from milk virtually in all dairy herds, the epidemiology and dynamics of environmental streptococcal mastitis and the effects on milk yield and milk quality are poorly documented. According to Ramanauskienė et al. (2008), Fatma et al. (2019) and Gezehagn et al. (2020) the most frequently isolated causative agents of bovine mastitis are streptococci (41.59%) and staphylococci (20.35%)

On the other hand, mastitis is one of the major causes of antibiotic use in dairy cows. There is a variety of antimicrobials that are used for the prevention and treatment of mastitis. Therefore, resistance to antimicrobials is expected. Resistance of mastitis pathogens to antimicrobial agents is another well-documented challenge in dairy industry (Bradley 2007; Päivi Rajala-Schultz et al., 2021).

Several trials for development of bovine mastitis vaccine against the most common contagious and environmental pathogens have been reported (Amorena et al., 1994; Hwang et al., 2000; Dosogne et al., 2002). Also, numerous attempts of vaccination have been made employing live S. aureus strains, Staphylococcus peptidoglycan, toxoid, or preparation of killed S. aureus, Streptococcus species and avirulent E. coli strains. Most of these vaccines increased the rate of spontaneous recovery from the infection and lessened its severity but in many cases did not prevent the reoccurrence of new infections (Monaci et al., 2015).

Because the only available control measure of bovine mastitis is the use of expensive antibiotics and commercially available vaccines, the main objective in the present work was to prepare an inactivated polyvalent autogenous vaccine against the most prevalent bacterial pathogens recovered from clinical and subclinical mastitis cases in a dairy farm and to evaluate its immunizing efficacy and therapeutic potential.

Material and Methods

Samples

Milk samples: A total of 3608 quarter’s milk samples were aseptically collected from 902 Friesian cows. The first few streams of milk were discarded and 15 to 20 ml of milk was collected separately from the four quarters of the udder into a sterile screw capped bottle. The collected milk samples were subjected to bacteriological examination. Also, the somatic cell count and total immunoglobulins level were determined (Olde et al., 2007).

Blood samples: Blood samples were collected from cows in all groups before immunization and at 2 weeks intervals post immunization and/or antibiotic treatments. Serum was separated and kept frozen till examined.

Preparation of milk whey

Five mg of rennin were dissolved in 270 ml of sterile saline and one ml of this solution was added to 10 ml of defatted milk sample. After 30 min incubation at 37ºC, the milk was centrifuged at 1000 xg for 20 min and then the supernatant (milk whey) was separated and kept frozen till examined (Frost and Tina, 1988).

Bacteriological examination

A loopful from the sediment of examined milk samples (pre-incubated at 37°C overnight and centrifuged at 3000 xg for 20 minutes) was streaked onto nutrient agar, blood agar and MacConkey agar plates. All plates were incubated at 37°C for 24-72 hrs and examined daily for bacterial growth. Single colonies were picked up and each was plated onto Edwards medium, Mannitol salt agar, EMB and Pseudomonas salt agar and incubated aerobically at 37°C for 24-72 hours for further identification. The isolated bacterial species were fully identified according to Quinn et al. (1994) and David et al. (2001).

Serotyping of the recovered bacterial isolates:

The recovered E. coli isolates were serotyped using commercial Escherichia coli antisera (Welcome Diagnostic Antisera). The isolated streptococcal species were serotyped using diagnostic reagent range from Oxoid (10 t/ch – P. 9939). These Kits were used for identifying Lancefield groups A, B, C, D and G of isolated streptococci. Dry spot kit (Staphytech plus - Oxoid. DR 100 M), and Latex slide agglutination test was used for identification of staphylococci.

Preparation of an inactivated polyvalent bovine mastitis bacterial vaccine: Bacterial species used: The following bacterial species, which were the most prevalent cause of mastitis in the examined dairy farms, were selected for preparation of an inactivated polyvalent autogenous mastitis vaccine. These include S. aureus, Str. agalactiae, Str. dysgalactiae and E. coli (serotype O111: K58 (B5).

Preparation of the bacterial antigen mass from obtained isolates: Briefly, the used bacterial species were separately inoculated into brain heart infusion broth and incubated at 37°C for 18 hours. The bacterial cells were harvested by centrifugation at 1800 xg for 20 min at 4°C and the supernatant was aseptically discarded. The cell sediment from these bacterial species was washed 3 times with sterile PBS and finally re-suspended in sterile PBS.

In the prepared polyvalent vaccine, the concentration of the bacterial components per vaccine dose was adjusted to 1010 CFU/5 ml of S. aureus, 4 x109 CFU/5 ml of Str. agalactiae, 4 x109 CFU/5 ml of Str. dysgalactiae and 1 x 109 CFU/5 ml of E. coli serotype O111 using phosphate buffer saline (Hogan et al., 1995; Jose et al., 1997).

Inactivation of the bacterial cells using gamma irradiation:

The bacterial mass of each of the 4 bacterial species was subjected to different doses of gamma irradiation trials to determine the minimum lethal dose according to Alur et al. (1998) and Ito (1998). The Minimum lethal dose of ionizing irradiation for the studied pathogens was found to be 4 kGy/min. The inactivated bacterial antigenic mass of the 4 included species in the abovementioned concentrations were mixed and freeze dried. The inactivated bacterial cells were mixed with Montanide, ISA-206 (SEPPIC, France) in a ratio 30:70 with continuous mixing Vincent et al. (2017). The mixture was gently dispersed using homogenizer till a stable oil emulsion with low viscosity was obtained.

Testing for sterility of the prepared vaccine: The vaccine preparations were inoculated on different bacteriological media, incubated for 3 days at 37°C and examined for the presence of any microbial growth (Vincent et al., 2017).

Vaccine efficacy trial

The prepared vaccine was evaluated in 6 groups of lactating cows (4 cows /group):

Experiment No. (1): It includes the first two groups which were clinically apparently normal cows and were used in evaluation of the immunizing efficacy of the prepared vaccine. (Vincent et al., 2017). The first group was kept unvaccinated and served as a control allover the study. The second group was immunized with the prepared vaccine.

Experiment No. (2): The other four groups of cows were included in this experiment. The cows in these 4 groups were suffering from subclinical mastitis and were used for evaluation of the therapeutic potential of the prepared vaccine 2ml /dose (Piepers et al., 2017). The first group (G1) was left untreated (neither antibiotics nor the vaccine). The second group (G2) was treated only with antibiotics (neomycin and penicillin). The third group (G3) was immunized with the prepared vaccine alone. The last group (G4) was treated simultaneously with both antibiotics and the prepared vaccine.

The vaccinated cows received two vaccinal doses at 2 weeks

 

Table 1: California mastitis test results for determination of the frequency of clinical and subclinical mastitis in dairy cows and percentage of quarters involvement.

Total numbers of cows examined	Clinical condition	Number of affected animals	%*	Total No. of quarters examined	Number of quarters affected	%
902	Animals with clinical mastitis	68	7.54	272	83	30.5
	Animals with subclinical mastitis	120	13.3	480	128	26.67

* Percentage was calculated according to the total number of examined cows.

 

Table 2: Incidence of pathogenic bacterial species in milk samples of cows with clinical or subclinical mastitis.

Isolated bacterial species	Clinical mastitis		Subclinical mastitis		Total number of isolates	%*
	No.	%	No.	%
E. coli	20	29.41	35	29.2	55	29.26
S. aureus	13	19.12	30	25	43	22.87
S. dysgalactiae	10	14.7	24	20	34	18.09
S. agalactiae	11	16.2	20	16.6	31	16.49
S. uberis	5	7.3	3	2.5	8	4.26
K. pneumoniae	3	4.4	4	3.3	7	3.72
P. aeruginosa	1	1.5	4	3.3	5	2.65
E. fecalis	3	4.4	0	0	3	1.60
S. pyogenes	2	2.9	0	0	2	1.06
Total	68		120		188	100

*Calculated in relation to the total number of isolates.

 

Table 3: Types, number and percentage of E. coli serotypes recovered from milk samples of cows suffering from clinical and subclinical mastitis.

E. coli serotypes	Animals with clinical mastitis		Animals with subclinical mastitis		Total
	No.	%	No.	%	No.	%
O111:K58 (B4)	9	45	12	34.3	21	38.2
O127:K63 (B8)	7	35	8	22.8	15	27.3
O 55:K59 (B5)	3	15	7	20.0	10	18.2
O125:K70 (B15)	1	5	5	14.3	6	10.9

 

Table 4: Total immunoglobulins (gm/dl) in serum and milk whey of apparently healthy cows immunized with the inactivated polyvalent mastitis vaccine.

Dairy cattle groups	Immunoglobulin's concentration (gm/dl) post immunization
	In serum		In milk whey
	2 weeks	4 weeks	2 weeks	4 weeks
Control (unvaccinated Cows)	1.48 ± 0.012	1.56 ± 0.034	2.83 ± 0.034	2.88 ± 0.043
Vaccinated cows	1.57 ± 0.036*	1.80 ± 0.049*	3.15 ± 0.018*	3.31 ± 0.030*

* Significant at P < 0.01 using t-test as compared with control.

 

interval and the vaccine was injected subcutaneously in the brachiocephalic muscle.

Blood and milk samples from cows in all groups were collected before and at 2 week interval post immunization and/or antibiotic treatment. Cows in all groups were monitored clinically allover the period of the study.

Statistical analysis

The obtained data were statistically analyzed using t-student test and One-way ANOVA according to Petrie and Watson (1999).

Results

A total of 3608 quarter’s milk samples were aseptically

 

Table 5: ELISA readings (OD) and corresponding titer of specific antibacterial antibodies in serum and milk whey of apparently healthy dairy cows immunized with the inactivated polyvalent bacterial vaccine.

		Reading	Control Non immunized Group (C.O.V.)*	Immunized apparently healthy cows (G I)
				E. coli	S. aureus	Str. agalactiae	Str. dysgalactiae
Serum samples	2 weeks	OD	0.465	0.584	0.615	0.579	0.499
		Titer	0	1/160	1/160	1/160	1/80
	4 weeks	OD	0.462	0.642	0.662	0.656	0.534
		Titer	0	1/320	1/640	1/320	1/160
Milk whey samples	2 weeks	OD	0.415	0.640	0.618	0.557	0.508
		Titer	0	1/160	1/160	1/160	1/160
	4 weeks	OD	0.411	0.752	0.641	0.595	0.539
		Titer	0	1/640	1/320	1/320	1/320

Results were expressed as OD and representative antibody titer.

* C.O.V. = mean of reading (OD) of control groups + 2 x S.E.

collected form 902 Friesian cows, of which 68 animals (7.54%) suffered clinical mastitis and 120 cows (13.3%) proved to suffer subclinical form of mastitis using California mastitis test (Table 1).

Bacteriological examination of collected milk samples from cows with clinical and subclinical forms of mastitis:

Several bacterial species were recovered from the examined milk samples (Table 2). The most prevalent bacterial species were E. coli (29.26%), S. aureus (22.87%), Str. dysgalactiae (18.09%) and Str. agalactiae (16.49%). Several other bacterial species were recorded at a lower rate including Str. uberis, K. pneumoniae, P. aeruginosa, E. fecalis and Str. pyogenes. Among the isolated E. coli isolates, the serotype O111:K58 (B4) was the most prevalent (38.2 %) (Table 3).

Experiment No. (1)

The immunizing efficacy of the prepared polyvalent inactivated mastitis vaccine in apparently healthy lactating cows:

Clinical signs and post vaccination reaction: Cows vaccinated with the prepared polyvalent inactivated mastitis vaccine manifested a mild vaccination reaction at the injection site in a form of swelling that subsides within 4 - 6 days post vaccination. Also, mild rise in rectal temperature (from 0.5 to 1ºC) of the injected animals was recorded one hour after the time of injection and remained for 2 days (data not shown). There was no significant reduction of milk yield following immunization with the prepared vaccine. All cows in these groups did not develop clinical mastitis during the whole observation period that extends up to 20 weeks post vaccination.

Immunoglobulin concentration: As shown in Table 4, significant (P < 0.01) increase in immunoglobulins concentration (g/dl) was recorded at the 4th week post immunization of the apparently healthy cows, which reached 1.80 ± 0.049 and 3.31 ± 0.030 g/dl in serum and milk whey, respectively, as compared to 1.56 ± 0.034 and to 2.88 ± 0.043 in serum and milk whey from the unvaccinated control cows.

ELISA results

As shown in Table (5), the highest level of antigen-specific antibodies against the immunogenic components of the prepared vaccine was recorded in serum and milk whey at the 4th week post immunization in cows treated with the vaccine alone. It reached 1/320 against E. coli (O111) and Str. agalactiae, 1/640 against S. aureus and 1/160 for Str. dysgalactiae, in serum while it reached 1/640 against E. coli and 1/320 against S. aureus, Str. agalactiae and Str. dysgalactiae in milk whey.

Experiment No. (2):

The therapeutic potential of the inactivated polyvalent bacterial mastitis autogenous vaccines in cows suffering from subclinical mastitis:

1. Effect of vaccination on the somatic cell count (SCC):

The analysis of SCC in milk samples collected before and 4 weeks post vaccination in cows suffering from subclinical mastitis is shown in Fig. (1). Significant (P < 0.01) reduction in the SCC after 4 weeks post vaccination and/or antibiotic treatment was recorded. In the antibiotic treated group the SCC decreased from 35 x 104 to 27 x 104. In the vaccinated group the SCC decreased from 34 x 104 to 24 x 104. In the third group that was treated with both the antibiotic and the vaccine, the SCC decreased significantly form 30 x 104 to 18 x 104.

2. Effect of vaccination on the immunoglobulin concentration: As shown in Fig. (2) and (3), significant (P < 0.01) increase of total immunoglobulins in the serum and milk whey was recorded in cows immunized with the prepared vaccine alone. In non-immunized cows and in cows treated with the antibiotics or with the vaccine and antibiotics no significant (P > 0.05) increase of total globulins in serum and milk whey was recorded.

Discussion

Despite the implementation of standard mastitis control programs, mastitis is still economically among the most important diseases of dairy animals. Approaches to enhance the cow’s immunity to prevent mastitis and thus minimize use of antibiotics have gained considerable attention. Yet, for a variety of reasons, vaccines developed for the prevention and control of mastitis have achieved only limited success. The multiplicity of pathogens capable of causing mastitis and insufficient knowledge of mammary gland immunology, bacterial virulence factors and mechanisms of pathogenesis are factors that have hindered development of effective mastitis vaccines. Several attempts have been made to develop and evaluate monovalent and polyvalent vaccines against the most prevalent mastitis pathogens like S. aureus, Str. agalactiae, E. coli and Str. uberis. In general, the commercially available S. aureus vaccines although have a limited to moderate ability to prevent new infection, they could enhance the spontaneous cure rates and reducing chronic infections (Piepers et al., 2012; Schukken, et al., 2014).

The first part of the present work was designed to determine the most prevalent bacterial pathogens that underline cases of clinical or subclinical mastitis in cattle. E. coli, S. aureus, Str. agalactiae and Str. dysgalactiae were the most common etiological agents recorded. Therefore, a custom designed inactivated polyvalent autogenous vaccine was prepared from these four species and its immunizing and immunotherapeutic potential was determined. The selected bacterial species were grown, mixed and inactivated using gamma radiation. The minimum inactivating irradiation dose was determined and proved to be 4 krad/min (kGy). Similarly, Hammad et al. (1998) reported that an irradiation dose of 4 kGy greatly reduced the microbial count and extend the shelf lifetime of foods. Variation among the intensity of irradiation doses (2-10 kGy) required for microbial inactivation has been reported by several authors (Cho et al., 1986; Leslie, et al., 2008; Fahmida et al., 2013, Wilmar et al., 2019; Eve et al., 2020). The variation of the intensity of irradiation required for inactivation was found to be influenced by several factors, among other, the microbial species involved and the nature of the irradiated material (solution vs. dried form) (Andreia et al., 2016; Yifan et al., 2018).

The immunizing and immunotherapeutic potential of the inactivated polyvalent bacterial mastitis autogenous vaccine were determined in a group of cattle in a dairy farm from which the above-mentioned bacterial species were recovered. Determination of the immunizing potential of the prepared vaccine was done in apparently healthy cows, while the immunotherapeutic efficacy was evaluated in cows suffering subclinical mastitis. Slight swelling at the site of the injection was observed in all vaccinated cows. It continued for 2-6 days post-injection and returned to normal after that. This swelling can be considered as a normal reaction to the injected vaccine. The vaccine was prepared using sterile distilled water free from pyrogenic and toxigenic elements. Compared with the control non-immunized cows, an increase of the rectal temperature (0.5 to 1 ºC) was noticed in the injected cows. It persisted for 2 days and became normal after that. The skin reaction and the elevation of temperature, however, can be attributed to the previous sensitization of the injected animals to one or more of the antigenic components of the used vaccine (Dosogne et al., 2002). No significant decrease in milk yield in the days immediately following the immunization was recorded.

The immunizing efficacy of the prepared vaccine was determined using two parameters; first the titer of the specific antibodies developed against microbial components of the vaccine and secondly the level of total immunoglobulins in sera and milk whey of the immunized cows. In the present work, evaluation of the vaccine was limited to its immunizing efficacy only, where the protective efficacy of the vaccine could not be determined. It was impossible for us to run intramammary experimental infection of the immunized cows.

Investigating the effect of immunization of cows with the prepared vaccine on the serum level of total immunoglobulins at 2- and 4-weeks post immunization revealed an increase in the immunoglobulins level in the immunized cows, which reached 1.57 and 1.80 g/dl as compared with 1.48 and 1.56 g/dl in control non-immunized group. Similarly, the level of total immunoglobulins in milk whey of the immunized cows reached to 3.15 and 3.31 g/dl at 2- and 4-weeks post immunization compared to 2.83 and 2.88 g/dl in the control non-immunized cows. Similar result was recorded by Gilman et al. (1991) and Tomita et al. (1998).

Immunization of cows with the prepared inactivated polyvalent bacterial mastitis vaccine variably stimulated the production of specific antibodies against the bacterial components of the vaccine both in the serum and milk whey. Two weeks post immunization a titer of 1/160 of antibodies specific to E. coli, S. aureus, Str. agalactiae and a titer of 1/80 against Str. dysgalactiae was measured in sera of the immunized cows. Four weeks post immunization (2 weeks after the booster dose) a significant increase was reported against S. aureus, where the antibody titer reached 1/640. Also increase in the anti- E. coli and anti- Str. agalactiae antibody titer reaching 1/320 was measured. An increase of 1/160 was recorded against Str. dysgalactiae. Similar results were reported by several authors using monovalent or polyvalent vaccine (Amorena et al., 1994; Zuhair, 2017). Also, Tomita et al. (1995 and 1998) and Hogan (1999) reported significant increase in IgG antibody titer against E. coli in serum of cattle vaccinated with E. coli j5 vaccine.

In the milk whey of the immunized cows, the anti-bacterial antibodies detected after two weeks from the immunization reached 1/160- and double-fold increase was measured two weeks following the booster dose. Similar results were reported by Tomita et al. (2000). However, it was observed that the level of anti E. coli antibodies was higher in milk whey than the level of anti S. aureus antibodies in contrast to what was recorded in the serum of the immunized cattle. This might be attributed to the immunoglobulin classes or subclasses produced against the different bacterial component of the vaccine and perhaps those produced against E. coli were of IgG1 isotype that are more secretable in milk compared to other immunoglobulin subclasses that are found in high concentration in blood (Amorena et al.,1994). Tomita et al. (1998) reported also that vaccination of heifers with S. aureus, Str. agalactiae and Str. uberis on days 55 and 45 prior to calving induced significant increase in the blood serum level of IgG1, slight increase in IgG2 content (as a percent of total immunoglobulins) and decrease in IgM content for 1 month after calving.

In spite of the fact that specific antibodies against the bacterial components of the vaccine was detected in blood and milk of immunized cows and none of the vaccinated cows developed clinical mastitis during the whole observation period (20 weeks), no clear statement could be concluded on the protective nature of the prepared vaccine. Evaluation of the protective efficacy required the intramammary experimental infection of cows with the pathogenic bacteria in an experimental model,

In the second experiment the immunotherapeutic potential of the prepared vaccine was evaluated in cows with diagnosed subclinical form of mastitis from which the vaccine components of bacterial species were isolated. All cows in the 3 treated groups did not proceed to clinical mastitis during the whole observation period that extended for 20 weeks. It was observed also that animals treated with the vaccine alone developed significant increase of specific antibodies against the microbial components of the used vaccine. However, in the group treated with the vaccine and antibiotics lower level of antibody response was measured. This might be attributed to possible immunosuppressive effect of the used antibiotics.

Analysis of the result of SCC determined 2 and 4 weeks post treatment revealed significant reduction after 4 weeks from start of the treatment in the three differently treated groups. However, in group that was treated with the antibiotic and the vaccine, the reduction of the SCC count was highly significant and the cell number returned to figures similar to those counted in the apparently normal controls. These results are in agreement with those reported by Jose et al. (1997).

Although the success of mastitis vaccine is difficult to define, but its use for therapeutic purpose proved effective as it stops the progress of subclinical cases to proceed to clinical mastitis and also was associated with reduction of the SCC to its normal level. Similar result was recorded by Hogan et al. (1995) who reported rapid recovery and reduction of clinical signs of experimental E. coli mastitis through vaccination.

Spontaneous recovery and significant reduction of the microbial CFU /ml of the examined milk samples was recorded in the present work. Leitner et al. (2004) reported that 30% of the S. aureus infected cows when vaccinated with a commercial S. aureus vaccine remained infection free till the end of 348-day trial, which suggest a curative role of this vaccine.

A recent study made by Athar (2007) demonstrated that locally prepared polyvalent bacterin mastitis vaccines (containing killed S. aureus, Str. agalactiae, and E. coli with various adjuvants) not only prevented new infections but also cured existing infections of these organisms in dairy buffaloes. Similar observations were made with locally prepared S. aureus vaccines (live attenuated vaccine, plain bacterin, dextran sulphate adjuvanted bacterin and oil-adjuvanted bacterin (Ahmed, 2010). Also Hwang et al. (2000) reported the therapeutic efficacy of autogenous S. aureus vaccines in Korea. They investigated therapeutic efficacy of an autogenous S. aureus toxoid-bacterin in lactating cows suffering from subclinical S. aureus mastitis and proved that the autogenous toxoid-bacterin treatment against S. aureus subclinical mastitis in lactating cows increase the cure rate of existing infections, reduce their severity and also prevent occurrence of the new infections. It is interesting to note that most S. aureus vaccines developed for the prevention of infection act as better curative than preventative agents (Clegg et al., 2021).

Conclusion

The obtained results indicated that the prepared polyvalent inactivated autogenous vaccine and the immunization program including subcutaneous injection of two vaccine doses, each of 2ml/animal at 2 weeks interval in the brachiocephalic muscle is efficacious in the reduction of the incidence of intramammary infection due to S. aureus, Coliforms, Str. agalactia and Str. dysgalactiae in cows. Also, immunization significantly reduces the severity of the symptoms and causes a significant increase in the spontaneous cure rate of cows suffering subclinical mastitis where no case developed acute mastitis.

Acknowledgments

This work is supported by the Science, Technology and Innovation Funding Authority (STDF) ID 27811.

conflict of interest

The authors declare no conflict of interest.

Novelty Statement

Institutional Animal Care and Use Committee at Central Laboratory for Evaluation of Veterinary Biologics approved the research manuscript and it has been reviewed under our research authority and is fulfilling bioethical standards.

authors Contribution

Experiments were designed by NGS and MSA, and the experiments were performed by HAK, NMA, and MSA.Data analysis was accomplished by NMA. ISY and MSA. The manuscript was written by HAK and MSA.

References

Ahmad T, Muhammad G, Yousaf A., Nadeem M, Zafar M.A (2010). Therapeutic Efficacy of Antibiotics and Bacterin-Toxoid in Sub-Clinical Streptococcus Agalactiae Mastitis in Dairy Buffaloes. Revist. Vet. 21 (1): 488-492.

Alur M.D, Kamot A.S., Doke S.N., Nair P.M (1998). “Development of indication process for eradicating salmonella and staphylococcus from pork meat products. J. Food Sci. Technol., 35 (1): 15-20.

Amorena B, Baselga R., Albzin I. (1994). Use of liposome-immunopotentiated expolysaccharide as a component of an ovine mastitis staphylococcal vaccine. Vaccine., 12 (3): 243-249. https://doi.org/10.1016/0264-410X(94)90201-1

Andreia I. Pimenta, Duarte Guerreiro, Joana Madureira, Fernanda M. A. Margaça, Sandra Cabo Verde (2016). Tracking Human Adenovirus Inactivation by Gamma Radiation under Different Environmental Conditions. Appl Environ Microbiol.; 82 (17): 5166–5173. Pre-published online 2016 Jun 17. Published online 2016 Aug 15. PMCID: PMC4988180. https://doi.org/10.1128/AEM.01229-16

Athar (2007). A preliminary study on the effect of inactivated polyvalent mastitis vaccines on milk quantity and quality in dairy buffaloes. Pakistan Vet. J., 27 (2): 85-91

Bradley AJ, Leach KA, Breen JE, Green LE, Green MJ Bradley AJ, Leach KA, Breen JE, Green LE, Green MJ.(2007). Survey of the incidence and aetiology of mastitis on dairy farms in England and Wales. Vet. Rec.; 24; 160 (8):253-7. https://doi.org/10.1136/vr.160.8.253

Calvinho; L.F.; Tarabla, H.D.; Canavesio, V.R.; Zubriggen, M.A.; Quaino, O. and De-Canavesio, V.R. (1989): Evaluation of a commercial live vaccine against Staphylococcus aureus mastitis. Vet. Arg., 6 (56): 380-383.

Cho H.O, M.W. Byun, J.H. Kwon, J.W. Lee, J.S. Yang (1986). Comparison of ethylene oxide (ED) and Irradiation treatment on the sterilization of spices. Korean J. Food Sci. Technol., 18: 283.

Clegg J, Soldaini E, McLoughlin RM, Rittenhouse S, Bagnoli F and Phogat S (2021) Staphylococcus aureus Vaccine Research and Development: The Past, Present and Future, Including Novel Therapeutic Strategies. Front. Immunol. 12:705360. https://doi.org/10.3389/fimmu.2021.705360

David E. Kerr, Karen Plaut, A. John Bramley, Christine M. Williamson, Alistair J. Lax, Karen Moore, Kevin D. Wells, Robert J. Wall (2001). Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice. Nat. Biotechnol., 19: 66 – 70. https://doi.org/10.1038/83540

Dosogne H, Meyer E, Sturk A, van Loon J, Massart-Leën AM, Burvenich C. (2002). Effect of enrofloxacin treatment on plasma endotoxin during bovine Escherichia coli mastitis. Inflamm Res., 51 (4): 201-5. https://doi.org/10.1007/PL00000293

Ebrahimi A, Nikookhah F, Nikpour S, Majiian F and Gholami M (2008). Isolation of Streptococci from milk samples of normal, acute and subclinical mastitis cows and determination of their antibiotic susceptibility patterns. Pak J. Biol. Sci., 11 (1): 148-50. https://doi.org/10.3923/pjbs.2008.148.150

Eve V Singleton, Shannon C David, Justin B Davies, Timothy R Hirst, James C Paton, Michael R Beard, Farhid Hemmatzadeh, Mohammed Alsharifi (2020). Sterility of gamma-irradiated pathogens: a new mathematical formula to calculate sterilizing doses. J. Radiat. Res., 61 (6): 886–894. Published online 2020 Sep 15. PMC7674690 https://doi.org/10.1093/jrr/rraa076

Fahmida BA, Kazi TA, S. M. Asaduzzaman, Kazi NH (2013). Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane. Biomed. Res. Int., PMC3770025- PMID: 24063009 https://doi.org/10.1155/2013/586561

Fatma M. Abo Zaid, Hassan Y. Abd ElHamed, El-Sagher O. Ahmed, Amal M. Eid, Adel E. A. Mohamed (2019): Frequently Isolated Bacteria From Lactating Cows With Clinical And Subclinical Mastitis In Gharbia Governorate, Egypt. Ass. Univ. Bull. Environ. Res. 22(1): 83-93. https://doi.org/10.21608/auber.2019.99900

Ferguson JD, Azzaro G, Gambina M, Licitra G. (2007): Prevalence of mastitis pathogens in Ragusa, Sicily, from 2000 to2006. J. Dairy Sci.; 90 (12): 5798-813. https://doi.org/10.3168/jds.2006-903

Frost A.J., Tina M. (1988). The effect on inflammation of the bovine mammary gland of vaccination with a cell wall extract of Staphylococcus aureus. J. Vet. Med., B35: 688-694. https://doi.org/10.1111/j.1439-0450.1988.tb00545.x

Gezehagn K, Betelihem T, Belege T (2020). Isolation and Identification of Major Pathogenic Bacteria from Clinical Mastitic Cows in Asella Town, Ethiopia. Vet Med Int.,  6656755. Published online 2020 Nov 13. https://doi.org/10.1155/2020/6656755, PMCID: PMC7683167, PMID: 33274039.

Gilman A.G., Rall T.W., Nies A.S., Taylor P. (eds) (1991).Goodman and Gilman’s: The Pharmacological Basis of Therapeutics.” 8th Ed., Pergamon Press, Member of Maxwell MacMillan Pergamon Publishing Corporation New York, Oxford, Beijing, Frankfurt, Sydney, Toronto.

Hammad A.A.L, T.M. El-Mangy, A.K Mabrouk (1998). Shelf-life extension and improvement of the microbiological quality of fresh sausage with irradiation.” 4th Arabic Conference on the Peaceful uses of Atomic Energy.

Hogan J.S., Weiss W.P., Smith K.L., Todhunter D.A., Schoenberger P.S., Sordillo L.M. (1995). Effects of Escherichia coli J5 vaccine on mild clinical coliform mastitis. J. Dairy Sci., 78 (2): 285-290. https://doi.org/10.3168/jds.S0022-0302(95)76636-X

Hogan J.S., Bogacz V.L., Aslam M., Smith K.L. (1999): Efficacy of an Escherichia Coli J5 bacterin administered to primigravid heifers. J. Dairy Sci., 82 (5): 939-943. https://doi.org/10.3168/jds.S0022-0302(99)75312-9

Hogeveen H, Huijps K, Lam TJ (2011). Economic aspects of mastitis: new developments. N Z Vet. J., 59: 16–23. https://doi.org/10.1080/00480169.2011.547165

Hwang CY, Pak SI, Han HR. (2000). Effects of autogenous toxoid-bacterin in lactating cows with Staphylococcus aureus subclinical mastitis. J. Vet. Med. Sci., 62 (8): 875-80 https://doi.org/10.1292/jvms.62.875.

Ito H. (1998). Irradiation effect of Escherichia coli O157:H7 in meats. Shokuhin-Shoska, 33 (1 -2): 29-32. https://doi.org/10.5986/jrafi.33.29

Jaime R, Efraín B, Carlos M (2018). Assessing Financial Impacts of Subclinical Mastitis on Colombian Dairy Farms. Front Vet. Sci., 5: 273. https://doi.org/10.3389/fvets.2018.00273, PMCID: PMC6277785 PMID: 30542654.

Jose A. Giraudo, Horacio, Alberto, Giraudo (1997). Field trials of vaccine against bovine mastitis. J. Dairy Sci., 80: 845-853. https://doi.org/10.3168/jds.S0022-0302(97)76006-5

Khaitsa ML, Wittum TE, Smith KL, Henderson JL and Hoblet KH (2000). Herd characteristics and management practices associated with bulk-tank somatic cell counts in herds in official Dairy Herd Improvement Association programs in Ohio. Am J Vet Res., 61 (9): 1092-8. https://doi.org/10.2460/ajvr.2000.61.1092

Leitner, U. Merin, N. Silanikove (2004). Changes in Milk Composition as Affected by Subclinical Mastitis in Goats G. American Dairy Science Association, J. Dairy Sci., 87: 1719–1726 https://doi.org/10.3168/jds.S0022-0302(04)73325-1

Leslie A. Dauphin, Bruce R. Newton, Max V. Rasmussen, Richard F. Meyer, Michael D. Bowen (2008). Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays. Appl. Environ. Microbiol., 74 (14): 4427–4433. Published online 2008 May 30. https://doi.org/10.1128/AEM.00557-08

Monaco C., Nanchahal J., Taylor P., Feldmann M. (2015). Anti-TNF therapy: past, present and future. Int. Immunol. 27, 55–62. https://doi.org/10.1093/intimm/dxu102

Mørk T, Waage S, Tollersrud T, Kvitle B, Sviland S (2007). Clinical mastitis in ewes; bacteriology, epidemiology and clinical features. Acta Vet. Scand., 24; 49:23. https://doi.org/10.1186/1751-0147-49-23

Ogola H, Shitandi A, Nanua J (2007). Effect of mastitis on raw milk compositional quality. J Vet Sci., 8 (3):237-42. https://doi.org/10.4142/jvs.2007.8.3.237

Olde Riekerink RG, Barkema HW, Stryhn H. (2007). The effect of season on somatic cell count and the incidence of clinical mastitis. J. Dairy Sci., 90 (4):1704-15. https://doi.org/10.3168/jds.2006-567

Päivi Rajala-Schultz, Ane N, Tariq H, Karin PW (2021). Prudent Use of Antibiotics in Dairy Cows: The Nordic Approach to Udder health. Front. Vet. Sci., volume 8, 1-7. Sec. Vet. Epidemiol. Econ. https://doi.org/10.3389/fvets.2021.623998.

Petrie A., Watson P. (1999). Statistics for Veterinary and Animal Science.” 1st Ed., pp. 90-99, The Blackwell Science Ltd, United Kingdom.

Piepers S., K. Deberdt, A. DeVisscher, J. Verbeke, S. De Vliegher (2012). Immunological response to an experimental intramammary inoculation with a killed Staphylococcus aureus strain in vaccinated and non-vaccinated lactating dairy cows. World Buiatrics Congress. June 5, Lisbon, Portugal. Pages 8-11. https://doi.org/10.3920/978-90-8686-742-4_68

Piepers S., A. Prenafeta, J. Verbeke, A. De Visscher, Ricard March, S. De Vliegher (2016). Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine J. Dairy Sci. 100:769–782 https://doi.org/10.3168/jds.2016-11269 © American Dairy Science Association®,

Quinn P. J., Carter M. E., Markey,B. K., Carter G. R. (1994). Clinical veterinary microbiology.” Mosby year book Europe limited, lynton House, London, PP. 109-126.

Ramanauskienė J., Sederevičius A., Aniulis E., Rudejevienė J., Želvytė R., Monkevičienė I., Laugalis J., Kabašinskienė A., Makauskas S. and Savickis S. Karvių klinikinio mastito gydymo efektyvumo tyrimai (2008): Vet Med Zoot.. T. 41 (63). P. 80−85.

Schlegelová J, Babák V, Klímová E, Lukásová J, Navrátilová P, Sustácková A, Sedivá I, and Rysánek D. (2002): Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples. J Vet Med B Infect Dis Vet Public Health, 49 (5): 216-25. https://doi.org/10.1046/j.1439-0450.2002.00520.x

Schukken Y.H., V. Bronzo, C. Locatelli, C. Pollera, N. Rota, A. Casula, F. Testa, L. Scaccabarozzi, R. March, D. Zalduendo, R. Guix, P. Moroni. (2014). Efficacy of vaccination on Staphylococcus aureus and coagulase-negative staphylococci intramammary infection dynamics in 2 dairy herds. J. Dairy Sci. 97: 5250-5264. https://doi.org/10.3168/jds.2014-8008

Tomita G.M., Todhunter D.A., Hogan J.S., Smith K.L. (1995). Immunization of dairy cows with an Escherichia coli J5 lipopolysaccharide vaccine. J. Dairy Sci., 78 (10): 2178-2185 https://doi.org/10.3168/jds.S0022-0302(95)76845-X.

Tomita G.M., Nickerson S.C., Owens W.E., Wren B. (1998). “Influence of route of vaccine administration against experimental intramammary infection caused by Escherichia coli. J. Dairy Sci., 81 (8): 2159-64. https://doi.org/10.3168/jds.S0022-0302(98)75793-5

Tomita G.M., Ray C.H., Nickerson S.C., Owens W.E., Gallo G.F. (2000). A comparison of two commercially available Escherichia coli J5 vaccines against E. coli intramammary challenge. J. Dairy Sci., 83 (10): 2276-2281. https://doi.org/10.3168/jds.S0022-0302(00)75112-5

Vincent H, Christophe G, Guillaume T, Maryline R, Laurine F, Christian T, Florence B. Gilbert, Edouard G, Céline B, Christophe S, David G. E. Smith, Pierre G, Gilles F, Pascal R (2017). Local immunization impacts the response of dairy cows to Escherichia coli mastitis. Scient. Rep. volume 7, Article number: 3441 https://doi.org/10.1038/s41598-017-03724-7

Wilmar C, Julius B, Jochen B, Lena H, Norbert R, Laura P, Dominik S, Kerstin S, Guillermo Martinez-de-Tejada, Klaus B, Thomas G (2019). Inactivation of Bacteria by γ-Irradiation to Investigate the Interaction with Antimicrobial Peptides. Biophys. J., 117 (10): 1805–1819. Published online 2019 Oct 18. https://doi.org/10.1016/j.bpj.2019.10.012

Yifan Z, Ralf M, Sophia T, Barbora D, Georgios A, Nicolas M, David D, Alexander M (2018). Geobacillus and Bacillus Spore Inactivation by Low Energy Electron Beam Technology: Resistance and Influencing Factors Front Microbiol., 9: 2720. Published online 2018 Nov 23. https://doi.org/10.3389/fmicb.2018.02720

Zadoks RN, Middleton JR, McDougall S, Katholm J, and Schukken YH.(2011). Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans. J. Mammary Gland Biol. Neoplasia., 16 (4):357-72 https://doi.org/10.1007/s10911-011-9236-y.

Zuhair BI (2017). Mastitis vaccines in dairy cows: Recent developments and recommendations of application. Vet. World., 10 (9): 1057–1062. Published online 2017 Sep 12. https://doi.org/10.14202/vetworld.2017.1057-1062