Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica and the Antimicrobial Effect of Plantago lanceolata Extract

H. A. Abdul-Ratha1*, Thaar Mohammed Najim2, Yaman Kahlid Sadeq1

1Al-Farahidi University, College of Science, Iraq; 2University of Fallujah, Biotechnology and Environmental Center, Iraq.

Received | July 29, 2025; Accepted | October 02, 2025; Published | October 15, 2025

*Correspondence | Hassan A. Abdul-Ratha, Al-Farahidi University, College of Science, Iraq; Email: [email protected]

Citation | Abdul-Ratha HA, Najim TM, Sadeq YK (2025). Isolation and identification of Pasteurella multocida and Mannheimia haemolytica and the antimicrobial effect of Plantago lanceolata extract. J. Anim. Health Prod. 13(s1): 607-615.

DOI | https://dx.doi.org/10.17582/journal.jahp/2025/13.s1.607.615

ISSN (Online) | 2308-2801

Copyright: 2025 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

The Pasteurella multocida are small, Gram-negative rods or coccobacilli bacteria and occur singly, or in pairs or short chains, bipolar staining may be seen and capsules may be present, they are non-motile, non-sporing, grow on MacConkey agar (Quinn et al., 2004; Momin et al., 2011; Dabo et al., 2007; Hala et al.,2020). Mannheimia haemolytica (formerly known as: Pasteurella haemolytica) has been the subject of extensive reclassification in the past: first called Bacterium bipolare multocidum by Zecchinon et al. (2005), it wasrenam ed Pasteurella haemolytica in 1932 (Zecchinon et al., 2005). The nomenclature of the diseases associated with infections with Pasteurella spp. in farm animals has been indefinite and confusing. A suggested nomenclature is based on the clinical findings and on the bacteria that are commonly associated with each entity (Radostits et al., 2010). Therefore, there are three main diseases caused by P. multocida and M. haemolytica The first disease form is septicemic pasteurellosis of cattle (hemorrhagic septicemia or barbone), commonly associated with infection by P. multocida type 1 or B, is the classical disease of southern Asia, while the second disease is pneumonic pasteurellosis of cattle, which commonly associated with infection by M. haemolytica biotype A serotype 1, and P. multocida biotype A, is a common disease in Europe and the western hemisphere. M. haemolytica tends to cause a fulminating fibrinous lobar pneumonia, and P.multocida causes a fibrinopurulent bronchopneumonia, whereas the last disease called pasteurellosis of pigs, sheep, and goats, in pigs and this is usually associated with infection by P. multocida and is mainly pneumonic in form, pasteurellosis of sheep and goats is usually associated with infection by M. haemolytica. Although it is often pneumonic in form, a septicemic form of the disease is not unusual, especially in lambs (Sayyed et al., 2024).

The study on the distribution of the isolates in relation to the health status of the examined sheep, 7.45% (n = 7) and 29.6% (n= 16) were isolated in the asymptomatic and symptomatic sheep, respectively indicating that the isolation rate of the bacteria is more than double in the symptomatic sheep as compared to that of the asymptomatic sheep, and from the total of 66 young aged animals (≤ 6 months) a total of 10 (15.15%) and 2 (3.03%), Manhaeimia haeimolytica and Pasteurella multocida species were isolated respectively, both of which accounted 52.2% (12/23) of the total isolates showing relatively greater proportion (except for P. multocida) as compared to the older age category (>6 months of age) in which 9.76% M. haemolytica, 3.66% P. multocida and 47.8% of the total isolates recovered (Fentahum and Tsegaw 2023). Plantago lanceolata, narrow-leaved plantain is one of over 250 species of genus Plantago L. (Plantaginaceae). It is common in roadside grassland, with a world wide distribution, perennial, rarely annual or biennial plant, with a mass of surface fibrous roots (5–10cm long) and often a few deep roots, and is wind-pollinated (Hassemer, 2019; Hassemer et al., 2019; Drava et al., 2019).

This plant is mostly grown in grasslands, rarely in arable land, and its secondary metabolites are used in medicine (Hassemer, 2019). It is very rapid growth and deep rooting in the soil, which results in high drought tolerance and uptake of valuable nutrients from deep soil layers An intensive symbiosis with various mycorrhizal fungi is characteristic of plantain for a high capacity for nutrient and water appropriation (Michal et al., 2021). The use of P. lanceolata is discussed on permanent and non-permanent grasslands where agriculturally specific varieties have been developed for grazing animals showing positive health effects in them (Michal et al., 2021).

The unusual properties were also observed in veterinary medicine, Studies showed the possibility of using this plant as an additive to rainbow trout (Oncorhynchus mykiss) feed, after 90 days of basic diet with the addition of plantain’s methanol extract and fulfillment of required breeding and experimental criterias, positive health effects were noted (Olumi et al., 2011).

The present study was aimed to investigate the prevalence of Pasteurella multocida and M. haemolytica in nasal swab and blood sample collected from different animals (cattle, sheep and goat) and study the antibacterial effect of alcoholic extract of Plantago lanceolata leaves against these bacterial isolates and compare it with different antibiotic.

MATERIALS AND METHODS

Collection of the samples

A total of four hundred and fifty of blood samples and four hundred and fifty nasal swab samples were collected from flock of Veterinary Medicine College, Baghdad University, Flock of Veterinary Medicine College, Fallujah University, flock of Agricultural College, Baghdad University and flocks around Al-Fallujah city.

The blood samples were collected in test tube with anti- coagulant The nasal swabs samples were collected from 150 cattle, 150 sheep and 150 goat respectively with different age and sex and 15, 20 and 15 samples were taken from infected lung of slaughtered cattle, sheep and goats respectively in abattoir and transported in cool box to the laboratory in order to use for bacterial isolation.

Plant material

Fresh Plantago (Plantago lanceolata) leaves were collected from a garden of Veterinary Medicine College, Baghdad University, which were classified in the Agriculture College, Baghdad University.

Pasteurella isolation

Bacterial isolation

Two methods were used to isolate Pasteurella from collected samples as follow:

Direct method

The samples of blood and nasal swabs were inoculated directly on the blood agar containing 5-7% sheep blood for recording the growth and type of hemolysis, samples were plated also on nutrient agar and MacConkey agar and colonies were tested for Gram stain. All plates were incubated aerobically at 37°C and examined for growth at 24- 48 hour. Then bacterial growth was purified on the same media. Infected lung samples sterilized by hot spatula, then incise was formed by a sterile incisor and samples take were by loop and culture directly on nutrient agar, blood agar and MaCconky agar.

Indirect method

Nasal swabs samples were cultured on test tubes containing 5ml of nutrient broth and incubated at 37C˚ for 24-48 h, then plated on blood and MaCconky agar media. Blood samples: Five ml of blood samples were added to 25ml Tryptic soya broth, incubated at 37 oC for 24-48 h, then plated on blood agar, nutrient agar and MaCconky agar. Infected lungs: A small piece of infected lung were placed in nutrient broth and incubated at 37 o C for 24-48 h then plated on blood agar and MaCconky agar media (Quinn et al., 2004).

Microscopical examination of gram and giemsa stain

1. Gram stain: It was conducted according Quinn et al. (2004).
2. Bipolar stain: It was conducted according Baron and Feingold (1990).
3. Capsule stain: It was conducted according Baron and Feingold (1990).

Biochemical tests

1. Catalase test it was conducted according to Mcfaddin (2000)
2. Oxidase test it was conducted according to Forbes et al. (2002)
3. KOH test it was conducted according Baron and Feingold (1990)
4. Oxidation-Fermentation (O-F) test according Quinn et al. (2004)
5. Sugar fermentation test according Ronald and James (2006).
6. Urease test according to Colle et al. (1996).
7. Indol test according Macfadden (2000).
8. Motility test according to Colle et al. (1996).

Identification by the VITEK 2 system

The VITEK 2 system is a new fully automated system to provide rapid and accurate identification and susceptibility testing results for the most clinical isolates, Identification was made on the bases of biochemical reactions, and MIC determinations were made by applying an algorithm to the growth kinetics monitored by VITEK 2 system (Funke et al., 1998; Bassel et al., 1997; Graf et al., 2000). These procedures were performed according to the manufacturer’s instructions.

Preservation of bacterial isolates

All isolates after definitive identification were cultured on sterile brain heart infusion broth and glycerol 20 % and incubated at 37C for 24 hrs, then after the occurrence of turbidity was stored in a deep freezing (Quinn et al., 2004).

Antibiotic sensitivity test

Five colonies of the same morphological type which were diagnosed previously were selected from nutrient agar, and a loopful was transferred to a tube containing 5ml of nutrient broth. The inoculated broth was incubated at 37 oC for 18 hrs turbidity appeared. A sterile swab was dipped into the standardized suspension of bacteria and excess fluid was expressed by pressing and rotating the swab firmly against the inside of the tube above the fluid level. Then, the swab was streaked in three directions over the entire surface of the Muller Hinton-agar. The inoculated plates were allowed to dry for 5 minutes, then the discs of antibiotics were placed onto agar surface using a sterile forceps. after that the plates were inverted and incubated aerobically at 37C for 18-24 hrs (Quinn et al., 2004).

After incubation, the zones of inhibition were measured by using electronic vernea depending upon a zone of inhibition reported.

Collection and preparation of plant samples

Fresh Plantago lanceolata leaves were collected from a garden of Veterinary Medicine College, Baghdad University. Plant leaves were washed under tap water and then dried at room temperature at shade. The dried leaves were crushed by a laboratory blender. The plant classification was performed in the Ministry of Agriculture, State Board for seeds testing and certification SBSTC in Abu Graib, Baghdad.

Preparation of alcoholic extract of Plantago lanceolata

Organic solvents were used for the extraction of P. lanceolata leaves by using absolute ethanol (Ethyl alcohol) which was considered as very effective in extracting the active ingredients of the plant according tothe method described by (Effrain et al., 2000). This was performed by using soxhlet apparatus, which around bottom glass flask placed that fitted to an extraction unit. The extracting unit contained the solvent and cellulose (thumble) located inside it that contained the dry plant powder. A distiller unit was fitted on to the extraction unit. For condensation of Vapor solvent, 30 g of plant powder were placed inside the thumble and 300 ml of absolute ethanol were placed inside the flask. The extraction was carried out for 24 hrs. by heating temperature that kept the solvent at 50- 60 co until a clear and colorless solvent appeared in the extracting unit. After that, the extract was dried by using a rotary evaporator 40-45 co. The dry extract was placed in an incubator under 38-40 co for complete dryness. The final extract was kept frozen at -20 co until use.

Determination the activity of Plantago lanceolata in vitro

Agar well diffusion method

Agar well diffusion method was used to check the activity of plant extract in vitro (Kavanagh, 1972). To achieve this purpose, pure colonies of pasteurella multocida and Mannheimia hemolytica were selected .Such colonies were inoculated into 4 ml of nutrient broth and incubated for 2-8 hrs at 37co, the turbidity of inoculums compared with standardized MacFaralnd tube number (one) containing (1.5 ×108) cfu/ ml, with a sterile cotton swab, the inoculum was spread evenly on the surface of Muller Hinton agar in pertridish, A five wells were made in Muller Hinton agar plates using a sterile cork borer (6 mm), 0.1 ml of different concentrations of plant extract (100, 150, 200mg /ml) were poured in the three wells while the other two wells were filled with 0.1 ml of DMSO and with ethyl alcohol as a control, the plates were incubated up down at 37 co for 24 hrs. Three replicates were carried out for each concentration extract, the diameter of inhibition zone was measured and the average values were recorded. The results and standard errors mean values were tabulated (Mahmood et al., 1989).

Statical analysis

The data obtained were expressed as Mean and Standard errors (Mean±SE) and subjected to statistical analysis using one-way and 2-ways analysis of variance (ANOVA) for comparison between the different groups. Also post hoc test was used when appropriate to find the least significant differences (LSD) between groups and (P <0.05) was considered statistically significant. All statistical analyses were done using the Statistical Package for the Social Sciences program (SPSS) version 20 packages (Snedecor and Cochran, 1989).

RESULTS AND DISCUSSION

Percentage of infection with pasteurella

As shown in Table 1 the Pasteurella multocida was isolated from 2 out of 150 blood samples of cattle with infection rate of 1.33% and 1 out of 150 nasal swab of sheep with rate of infection of 0.66%. All other samples from different animal species were appeared to be free from this bacteria, The total infection rate with Pasteurella multocida were 0.66%. Mannheimia haemolytica was isolated from 3 out of 150 blood samples of cattle with infection percent of 2%, while all other samples of sheep and goats were not revealed infection with this bacterium (Table 1).

This rates of infection with P. multocida and M. haemolytica in the present study were less than what revealed in other studies, where Radostits et al. (2010) have mentioned that both morbidity and case-fatality rates vary between 50% and 100%. This decrement in the morbidity rate may be attributed to the use of vaccine against these bacteria in Iraq during the last years and the constant climate during the period of the study which lead to absent or less stress on animals of the study (Personal contact), This may also be due to adaptation of the animals to these bacteria because Iraq is endemic with pasteurellosis. The researchers found that morbidity depended on the immune status of the herd, either acquired naturally or induced by vaccination, the greater the percentage of immune to non-immune animals, the lower will be the morbidity and in endemic areas, adult animals developed a naturally acquired immunity and large outbreaks no longer occured in these areas (Radostits et al., 2010).

In Iraq, Mubarak (2000) has isolated M. haemolytica at a percent of 2.94% from different animals, this result in agreement with our result.

Isolation and identification of Pasteurellac

The identification of the bacteria was ensured according to the (Quinn et al., 2004) by the following steps:

Cultural characteristics

The isolated colonies of Pasteurella multocida appeared as grey and viscous, with a strong mucinous odour,rough, irregular and with no hemolysis on blood agar and have no ability to grow on MacConkey agar. These characters are in agreement with those mentioned by Quinn et al. (2004).

The colonies of Mannheimia haemolytica on blood agar appeared as small, and greyish colonies produced beta hemolysis and grown on MacConkey agar with pink colour. These characters are in agreement with those mentioned by Quinn et al. (2004).

 

Table 1: The prevalence level of Pasteurella multocida and M. haemolytica in nasal swab and blood samples from cattle, sheep and goats.

Sample		Psteurella multocida			Mannhaimia haemolytica
		Cattle	Sheep	Goat	Cattle	Sheep	Goat
Blood	450	2(1.33%)	0	0	3(2%)	0	0
Nasal swab	450	0	1(0.66%)	0	0	0	0
Lung	50	0	0	0	0	0	0
Total	950	2(1.33%)	1(0.66%)	0	3(2%)	0	0

 

Microscopical examination of gram stain

Gram stain and KOH test

Staining of suspected colonies with Gram stain showed a pleomorphic gram-negative coccobacillus which occurred singly, in pairs or short chains. These characters are in agreement with those mentioned by Jawetz et al. (2007). The results of Gram stain were confirmed by KOH test, where all the isolates of P. multocida and M. haemolytica in the present study revealed a positive result to KOH test. This result of KOH test were inversely related to the Gram stain result (Quinn et al., 2004).

Geimsa stain

Staining with Geimsa stain revealed the presence of colors only of two opposite poles of the bacterium and leaving the rest of the bacterium unstained or of a lighter color.

Results of biochemical test

The results of most important biochemical tests that were used to differentiate between P. multocida, and M. haemolytica and with other types of bacteria shown in Table 2.

The results of identification which were obtained by biochemical tests that mentioned above were confirmed by using VITEK 2 system for one isolate only (Table 3).

The results of the P.multocida diagnosis by Vitek 2 system showed that these bacteria were positive for some biochemical tests including Dmannose, Tyrosine Aryl Aminidase, ELL MAN, Coumarate and phosphatase andnegative for the remaining biochemical tests (Table 3).

Antibiotic susceptibility test

Antibiotic susceptibility test was conducted for 3 isolates of P. multocida and 3 isolates of M. hemolytica against different antibiotics to assess the prevalence of antibiotic resistance of each isolate that was tested against ten antibiotics and inhibition zone of bacterial growth around the antibiotic disc were measured to check the sensitivity of the isolate against these ten antibiotics. The results are shown in Table 4.

 

Table 2: The results of biochemical tests of the Pasteurella isolates.

Tests species

Cata-lase

Oxi-dase

O/F

Beta haemo-lysis

Motility

Growth on macconkey

Sugar fermentation

Indole

Urease

Glu-cose

Lactose

Sucrose

P. multocida

+

+

F

-

-

-

+

+

+

-

+

-

M. haemolytica

+

+

F

+

-

+

-

-

+

+

+

+

 

Table 3: The results of VITEK2 system.

APPA	-	ADO	-	PYrA	-	IARL	-	dCEL	-	BGAL	-
H2S	-	PNAG	-	AGLTp	-	dGLU	-	GGT	-	OFF	-
BGLU	-	Dmal	-	dMAN	-	dMNE	+	BXYL	-	BALap	-
ProA	-	LIP	-	PLE	-	TYrA	+	URE	-	dsOR	-
SAC	-	dTAG	-	dTRE	-	CIT	-	MNT	-	5KG	-
ILATK	-	AGLU	-	SUCT	-	NAGA	-	AGAL	-	PHOS	+
GLYA	-	ODC	-	LDC	-	IHISa	-	CMT	+	BGUR	-
OI29R	-	GGAA	-	IMLTa	-	ELLM	+	ILATa	-

 

Table 4: Antibiotic sensitivity test of each P. multocida and M. haemolytica to different antibiotics.

Antibiotics	P. multocida		M. haemolytica
	Sensitive (%)	Resistant (%)	Sensitive (%)	Resistant (%)
Penicillin	0	3(100)	2(66.7)	1(33.3)
Erythromycin	1(33.3)	2(66.7)	2(66.7)	1(33.3)
Gentamycin		0	3(100)	0
Aztreonam	2(66.7)	1(33.3)	1(33.3)	2(66.7)
Doxycycline	1(33.3)	2(66.7)	2(66.7)
Rifampin	0		2(66.7)	1(33.3)
Amoxicillin + Clavulanic	1(33.3)	2(66.7)	2(66.7)	1(33.3)
Clindamycin	2(66.7)	1(33.3)	3(100)	0
Colistin sulfate	2(66.7)	1(33.3)	0	3(100)
Cloxacillin	0	3(100)	0	3(100)

 

Results showed that, Gentamycin was found to be the most effective antibiotic with 100% for both P. multocida and M. haemolytica,this result were in agreement with Naz et al. (2012) who haves found that P. multocida were sensitive to Gentamycin, and disagree with her result and the result of Sabiel et al. (2011) who found that Gentamycin was resistant, but disagree with those result for M. heamolytica that were found resistant to Gentamycin, Erythromycin and Penicillin. The results of Krisztina et al. (2024) showed that Atotal of 131 (84.5%) strains of Pasteurella multocida were susceptible to penicillin, and the same number of strains proved to be susceptible to enrofloxacin while A 100% resistance rate was observed for clindamycin. For erythromycin, 81.3% of strains showed reduced sensitivity while 9.7% were resistant. The result of Marru et al. (2013) were in disagreement with our results, who Have found that both P. multocida and M. haemolytica were resistant to Gentamycin. A study of ShivaChandra et al. (2004) that tested twenty antibiotics against P. multocid found highly susceptible to doxycycline, and their results was incompatible with our results. Pasteurella multocida is a widespread facultative pathogenic bacterium, which causes a wide range of diseases in both mammals and birds, the antibiotic susceptibility of 155 P. multocida strains were tested using the broth microdilution method to obtain the minimum inhibitory concentration (MIC) values for 15 antibiotics, The most effective antibiotics against pasteurellosis were ceftiofur, tetracycline, doxycycline, florfenicol and tilmicosin. Of the strains, 12 proved to be multi-drug resistant (MDR) (Krisztina et al., 2024), some of these results were somewhat consistent with what we obtained. The present study showed that Erythromycin was not very effective antibiotic. These observations are in accordance with Watts et al. (1994) who have frequently encountered resistance of M. haemolytica and P. multocida of bovine origin to erythromycin. Our results were in agreement with Mohammadi et al. (2006) who concluded that M. haemolytica were sensitive to Penicillin and disagree with his result that P. multocida were resistant to Penicillin. The result of de Jong et al. (2014) showed that both P. multocida and M. haemolytica were susceptible to amoxicillin/clavulanic acid, and these finding were with our result for M. haemolytica and compatible with result of Nedbalcova et al. (2010), but disagree with our result for P. multocida which show resistant of this bacteria. Klima et al. (2014) have reported that both P. multocida and M. haemolytica were show resistant for to most antibiotics. Zaheer et al. (2012) demonstrated that both M. haemolytica and P. multocida were erythromycin resistance, and such result agree with our result that showed resistance of P. multocida, but disagree with the result of M. haemolytica sensitive.

Yield extract of Plantago lanceolata

The leaves of the Plantago lanceolata which were extracted with 95% ethanol yielded deep green extract with plant powder of 24.7 %. This was determined by using the following equation:

Percentage yield of the extract = weight of extract (gm)/weight of plantago powder (gm) × 100 (Banso and Adeyemo, 2006)

Example: 7.41 g /30 g × 100 = 24.7 %

The effect of Plantago lanceolata extract against the P. multocida and M. haemolytica growth in vitro

The results showed that the concentration of the extract played an important role in determining the diameter of inhibition zone, as the diameter was increased when a higher concentration of the extract was used. The mean of inhibition zone diameter of the alcoholic extract of the P. lanceolata against P. multocida were 17.22, 19 .11, 21.94 mm while were 13.00, 14.55, 15.55 mm against M. haemolytica at the concentration of 100, 150, 200 mg/ml of the extract, respectively as shown in Tables 5 and 6.

The low concentration (100mg /ml) showed a low inhibition zone of 17mm and a high inhibition zone of 18mm for Pasteurella multocida, while a low inhibition zone of 11mm and a high inhibition zone of 16mm in Mannheimia haemolytica. At concentration of 150 mg/ml a low inhibition zone was a 18mm and a high inhibition zone was 20 mm for Pasteurella multocida, but for Mannheimia haemolyticaa low inhibition zone was 14mm and a high inhibition zone was 15 mm. At a high concentration of (200mg /ml) the low inhibition zone was 19.5mm and a high inhibition

 

Table 5: Sensitivity (inhibition zone mm.) of P. multocida against different concentrations of Plantago lanceolata extract.

No. of isolate	Concentration			DMSO	Alcohol
	100 mg/ml	150mg/ml	200mg/ml
P1	17.00 0.00C	18.33 0.57B	19.83 0.28Ac	0.00 0.00D	0.00 0.00D
P2	17.00 0.00C	20.00 0.00B	23.66 0.57Aa	0.00 0.00D	0.00 0.00D
P3	17.66 0.57C	19.00 0.00B	22.33 0.57Cb	0.00 0.00D	0.00 0.00D
Total mean	17.22	19.11	21.94	0.00	0.00

 

P=Pasteurella multocida The different uppercase letters refer to significant differences between differentcolumn at (P<0.05). The different lowercase letters refer to significant differences between different row at (P<0.05).

 

Table 6: Sensitivity (inhibition zone mm) of M. haemolytica against different concentrations of Plantago lanceolata extract.

No. of isolate	Concentration			DMSO	Alcohol
	100 mg/ml	150mg/ml	200mg/ml
M1	11.33 0.57Cb	15.00 0.00Aa	14.00 0.00Bc	0.00 0.00D	0.00 0.00D
M2	16.00 0.00Aa	14.00 0.00Cb	14.66 0.57Bb	0.00 0.00D	0.00 0.00D
M3	11.66 0.57Cb	14.66 0.57Bc	18.00 0.00Aa	0.00 0.00D	0.00 0.00D
Total mean	13.00	14.55	15.55	0.00	0.00

 

M= Mannheimia haemolytica. LSD=0.55.

 

zone was 24mm for Pasteurella multocida while for Mannheimia haemolytica a low inhibition zone was 14mm and a high inhibition zone was 18 mm; but no inhibition zone was recorded for DMSO and alcohol. In this regard the study of Alla et al. (2023) showed that Different concentrations of the ethanolic extract of P. major leaves exhibited different zones of inhibition against P. aeruginosa from 9.93 mm to 22.18 mm in diameter.

The results were showed the superiority of the concentration 200 mg/ml of plant extracts which could be attributed to a high amount of soluble active ingredients which decrease or prevent the bacterial growth. The antibacterial actions of the P. lanceolata extract could be attributed to their metabolites such as flavonoides, phenol, tannins, saponins, steroids, glycosides and resins were characterized by their antibacterial actions (Cowan, 1999). Different concentrations of the ethanolic extract of P. major leaves exhibited different zones of inhibition against P. aeruginosa from 9.93 mm to 22.18 mm in diameter. The ethanolic extract of P. major was able to inhibit the growth of S. pyogenes ATCC 19615 at concentrations of 250 mg/mL, 500 mg/mL, and 750 mg/mL, and 1000 mg/mL. Based on the thin layer chromatography test, the ethanolic extract of P. major contains a class of flavonoid compounds, alkaloids, steroids, phenols, tannins, and terpenoids. The mechanisms of action of active ingredients of P. lanceolata to impair the bacterial growth are different, such as tannic acid which have the ability to precipitating the gelatin of solution in a process known as astringency. Therefore, precipitating the protein and thus deprive the bacteria from the nutritional proteins (Giessman, 1963). On the other hand, the phenolic compounds exert their antibacterial action through the hydroxyl group (OH-) (Cowan, 1999). The flavonoids which can be synthesized by plants in response to the microbial infections (Dixon et al., 1983) could be able to make a complex with the intracellular proteins and cell wall, and disrupting the lipophilic compounds of the bacteria (Chabot et al., 1992).

The substitution of the ester or carboxylic acid functional group on the coumarine ring gave it a potent inhibitory action against both gram positive and gram negative bacteria (Kawase et al., 2001; Kairat et al., 2023). The superiority of the use of the plant extract, compared with the antibiotics, could be attributed to ability of the plant extract to attenuate of the pathogen virulence by allowing the immune system to participate in the elimination process of the pathogen, while the bactericidal antibiotics kill the bacteria directly, thus the anti-pathogenic ingredients in the P. lanceolata prevented the production of toxins or abolished the bacterial capability to adapt the host environment and would give a competitive advantage to the host immune system for clearance of the pathogens (Gonzalez-Lamothe et al., 2009).

In addition to what mentioned above, the acidity of the alcoholic extract also inhibited the bacterial growth through their free hydrogen ions which banded to molecules and changed the microbial environment (Joklik et al., 1992). The biostatistics analysis revealed significant differences (P < 0.05) between P. multocida and M. haemolytic, where as P. multocida isolates appeared significantly more sensitive to P. lanceolata extract than M. haemolytica isolates.

ACKNOWLEDGEMENT

We want to dedicate thanks and appreciations to Microbiology Department, College of Veterinary Medicine, University of Baghdad, Iraq and Fallujah University, Iraq for their support during the course of this work.

NOVELTY STATEMENT

This study has proven the therapeutic importance of Plantago lanceolata extract with the in cooperation between authors and through their own efforts with the assistance of specialized laboratories at the university of Baghdad and Fallujah University.

AUTHOR’S CONTRIBUTION

HAA-R: Conceived of the presented idea, investigate and supervised the findings of this work and supervised the project.

TMN: Verified the analytical methods, carried out the experiment.

YK: Laboratory work assistant.

Generative AI and AI-assisted technology statement

The authors declare that no Genrative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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