Pathognomonic features of Pasteurella multocida isolates among various avian species in Sharkia Governorate, Egypt

The present study aimed to isolate Pasteurella multocida (P. multocida) from pulmonary cases in several avian species and then investigate the histopathological features, antimicrobial resistance determinants, virulence characteristics, and risk factors analysis of the isolates in each species in correlation with epidemiological mapping of pasteurellosis in Sharkia Governorate, Egypt. The obtained data revealed a total occurrence of 9.4% (30/317) of P. multocida among the examined birds (chickens, ducks, quails, and turkeys). The incidence rate was influenced by avian species, climate, breed, age, clinical signs, and sample type. Antimicrobial susceptibility testing revealed that all isolates were sensitive to florfenicol and enrofloxacin, while 86.6 and 73.3% of the isolates displayed resistance to amoxicillin-clavulanic acid and erythromycin, respectively. All of the P. multocida isolates showed a multiple-drug resistant pattern with an average index of 0.43. Molecular characterization revealed that the oma87, sodA, and ptfA virulence genes were detected in the all examined P. multocida isolates. The ermX (erythromycin), blaROB-1 (β-lactam), and mcr-1(colistin) resistance genes were present in 60, 46.6, and 40% of the isolates, respectively. Ducks and quails were the most virulent and harbored species of antimicrobial-resistant genes. These results were in parallel with postmortem and histopathological examinations which detected more severe interstitial pneumonia lesions in the trachea and lung, congestion, and cellular infiltration especially in ducks. Epidemiological mapping revealed that the Fakous district was the most susceptible to pasteurellosis infection. Thus, farmers are recommended to monitor their flocks for signs of respiratory disease, seek veterinary care promptly if any birds are sick, and avoid the random usage of antibiotics. In conclusion, this study presents a comprehensive picture of the risk factors in correlation to the pathognomonic characteristics of P. multocida infection in poultry sectors to help in developing more effective strategies for prevention and control. Supplementary Information The online version contains supplementary material available at 10.1007/s11274-023-03774-2.


Introduction
Infections with P. multocida frequently result in epidemics of fowl cholera, causing enormous financial losses for the global poultry sector (Sid et al. 2015).
The mechanism of disease occurrence in P. multocida infections is as follows: the bacterium is inhaled or ingested by the host, attaches to the host's cells, produces toxins that damage the host's cells and tissues, and evades the host's immune system, thus causing disease (Wilson et al. 2002).
acquire new genes through horizontal gene transfer when it encounters other bacteria, such as E. coli (Wilson et al. 1993;El-Demerdash et al. 2023a;Megahed et al. 2023).This can lead to the development of new more virulent and difficult to treat strains of P. multocida isolates.Multidrug resistance has been exacerbated worldwide, resulting in a public health threat.Several recent studies have reported the emergence of multidrug-resistant bacterial pathogens from different origins necessitating proper use of antibiotics.Routine application of antimicrobial susceptibility testing is necessary to detect the antibiotic of choice and to screen for the emerging MDR strains (El-Demerdash et al. 2018;Algammal et al. 2021Algammal et al. , 2022aAlgammal et al. , 2023;;Ebrahem et al. 2023).
Being aware of the pathognomonic features of P. multocida isolates is important for the development of new diagnostic tests, vaccines, and treatments for infections caused by this bacterium.This study aims to understand the characteristics and molecular pathogenesis of various P. multocida isolates circulating in chickens, ducks, quails, and turkeys providing a comprehensive investigation and epidemiological mapping of the prevalence and diversity of P. multocida among various avian species in Sharkia Governorate.

Ethical approval and sampling
With approval number ZU-IACUC/2/F/216/2023, the study was carried out with the approval of the Faculty of Veterinary Medicine, Zagazig University, in accordance with its rules.A total of 317 birds (153 chickens, 36 quails, 38 turkeys and100 ducks), apparently healthy, diseased and dead were randomly collected from seven different districts in Sharkia Governorate of different types of production sectors, breeds, and ages during the period between July 2022 and March 2023.Samples of dead birds were collected from the liver, heart, lung, trachea, brain, kidney, and spleen while tracheal swabs were collected from live ones and all samples were subjected to bacterial examination.The sample collection procedures according to Panna et al. (2015) were utilized.

Pathological examinations
The examinations were conducted on specimens from the tracheas and lungs of infected birds (chickens, ducks, quails, and turkeys) that had either been sacrificed or were freshly dead.The specimens were then fixed in 10% buffered neutral formalin and paraffin sections of 2-3-micron thickness were prepared and stained with hematoxylin and eosin.These sections were then examined microscopically (Suvarna et al. 2013).

Isolation and identification of P. multocida
The isolation procedures were conducted by scorching the surface of the organs with a hot spatula and then sterilizing loopfuls, and inoculating swabs onto Tryptone Soya Broth (TSB, OXOID, Hampshire, United Kingdom).The inoculated TSBs were then incubated aerobically for 24 h at 37 °C and streaked onto 5% sheep blood agar and MacConkey's agar (OXOID, Hampshire, United Kingdom) for 24 h incubation period at 37 °C.Pure colonies were recognized morphologically using the Gram stain, Leishman's staining method, and biochemical assays (catalase, oxidase, nitrate, methyl red, Voges-Proskauer, sugar fermentation, indole, citrate, gelatin liquefaction and urease) (Carter 1984;Markey et al. 2013).

Molecular confirmation and typing
These assays were conducted in the Biotechnology Unit, Animal Health Research Institute, Zagazig Branch, Egypt, following a manual previously published in Shalaby et al. (2021).
DNA was extracted from each isolate using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH Catalogue no.51304) and PCR amplification was performed using primers listed in Table 1.
The inhibition zones, in millimeters, were measured in duplicate and scored as sensitive, intermediate, and resistant categories following the critical breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI 2020).Isolates resistant to ≥ 3 different antimicrobial classes were classified as multidrug-resistant (MDR).The multiple antibiotic resistance (MAR) index for each isolate was calculated as the number of antimicrobials to which the isolate displayed resistance divided by the number of antimicrobials to which the isolate had been tested (Tambekar et al. 2006).The tested isolates were categorized as multidrug-resistant (MDR), extreme drug-resistant (XDR), and pan-drug-resistant (PDR) as described by Magiorakos et al. (2012).

Detection of virulence and antimicrobial resistance genes
Plasmid DNA was extracted from bacterial isolates using Thermo Scientific GeneJET Plasmid Miniprep Kit (Thermo, Germany, Catalogue no.K0503) for detection of antimicrobial resistant genes.
On a 1.5% agarose gel (Applichem, Germany, GmbH), the PCR products were separated by electrophoresis, and the gel was photographed using a gel documentation framework (Alpha Innotech, Biometra).The data was analyzed by computer software.As a quality control, P. multocida ATCC® 43137™ was utilized.

Statistics and data analyses
Chi-square or Fisher's exact test (as needed) were used to compare categorical variables and two-tailed, unpaired student t-test was used to compare numerical variables.In all statistics, p-value at 0.05 were used as cutoff level for significance.The statistics were done using base functions in the R software version 4.3.1.
Heat maps and hierarchical clustering was used to visualize the overall occurrence of analyzed traits in the isolates and the relation among them and was done using the Pheatmap package in r software version 4.3.1 (Kolde 2012).Before correlation analyses, variables were tested for normality using Q-Q plot.Pearson correlation was estimated and visualized using the Hmisc package in R software version 4.3.1 (Martins 2022).Significant correlation pairs were determined at p-value of 0.05.The frequencies of studied genes/phenotypes were plotted as stacked bar graph using ggplot package in R software version 4.3.1 (Wickham et al. 2009).
To determine the influence of various predictor variables on the infection outcome, we treated each avian species (e.g.chicken) as a stand-alone dataset.For each species, we run a univariate logistic regression model on the independent variables to select significant ones as they relate to infection outcome, then a backward selection of those significant was applied to run a multivariate logistic regression model.Since the effect of some predictors on the infection outcome varies according to other variables, we included an interaction term in the regression equation.These analyses yielded β-coefficient, odds-ratio, and p-value for each predictor.The analyses were done using glm function in R software version 4.3.1.

Epidemiological mapping for pasteurellosis infection among examined avian species
The final collected data were subjected to ArcGIS application for geo-mapping the rate of P. multocida infection through Sharkia governorate, Egypt.

Clinical signs
General respiratory disorders were observed in most of the examined infected birds.In turkeys, a mild swelling of the head with eye discharge was observed in some cases.Sneezing, depression, mucoid discharge from the mouth, ruffled feathers, increased respiratory rate, and diarrhea which was most common in chickens and quail, were observed.Ducks suffered from tracheal rales with extended neck and nasal discharge.High morbidity rates (42-86%) were detected, while the mortality rate was mild to moderate in the inspected farms (5-31%).Decreased feed intake and weight loss were detected with the progress of the disease, especially in cases of severe infection.

Postmortem examination
The prevalence of gross lesions based on the trachea and lung were the most affected organs exhibiting inflammation in variable severity degrees.Congestion was marked especially in ducks, and catarrhal tracheitis represented in the trachea was filled with yellowish exudates in other cases such as in quails and chickens.Mucosal hemorrhagic spots may appear in severe cases.Examined lungs from positive infected cases appeared in different forms, an edematous, firm with cut surface yielded blood-tinged exudate and mild to moderate hemorrhage of mostly focal distribution, focal to diffuse congestion.The incidence and prevalence of gross lesions based on the examined birds were summarized in the lesion score (Table 2).

Microscopical findings
Tracheal lesions were cystic dilation of some mucosal glands (Fig. 1A), partial to complete detached cilia with hemorrhage and cellular infiltration (Fig. 1B), destructed mucosa with severe extravasated erythrocytes (hemorrhage) (Fig. 1C), necrosis of some chondrocytes of tracheal cartilage (Fig. 1D), and complete and partial destruction of mucosa and tracheal glands with or without normal cartilage (Fig. 1E&1F), perivascular cellular infiltration and edema with congestion (Fig. 1G).Perivascular extravasated erythrocytes with vascular congestion, endotheliosis (endothelial cell degeneration), and atrophy of tracheal muscle fibers (Fig. 1H) were also observed, along with perivascular fibrosis with or without atrophy of tracheal muscle fibers (Fig. 1I).
Lung lesions were more prominent, especially in ducks and included pneumonia represented in diffuse congestion and cellular infiltration with variable degrees of severity (Fig. 2A), bronchopneumonia, mild to severe focal to diffuse interstitial hemorrhage with focal inflammatory cellular infiltration (Fig. 2B) of mostly neutrophile, macrophages, and perivascular cellular infiltration with focal partial alveolar stenosis (Fig. 2C).Perivascular oedema, and fibrosis with or without cellular infiltration (Fig. 2D) were also observed, along with perivascular fibrosis with extravasated erythrocytes (Fig. 2E), hyperplasia of bronchial epithelium accompanied bronchopneumonia in some cases (Fig. 2F) and partial focal emphysema only in some chronic cases (Fig. 2G).Interstitial extravasated erythrocytes (Fig. 2H) and congestion of blood vessels with perivascular oedema (Fig. 2I) were also detected.Vascular changes in the lung were common and more observed in most cases which were represented by vacuolation of vascular tunica media with perivascular edema and cellular infiltration (Fig. 3A), endotheliosis and perivascular fibrosis (Fig. 3B) and perivascular cellular infiltration (Fig. 3C).
Full coverage of all previously mentioned microscopical lesions in different examined birds was illustrated in Table 3.

The phenotypic characteristics of the recovered P. multocida isolate
The isolates were examined microscopically, they were Gram-negative bipolar coccobacilli, dew grey, drop-like mucoid, non-hemolytic on blood agar, and failed to develop on MacConkey agar.The recovered isolates tested positive for catalase, oxidase, indole production, nitrate reduction, d-glucose, d-mannitol, galactose, fructose, and sucrose fermentation, and negative for methyl red, Voges-Proskauer, urease, citrate utilization, and gelatin liquefaction.

The prevalence of P. multocida in various birds and breeds and relation to the locality, breeding type, and sampled organs
From 317 tested birds, a total of 30 (9.4%) P. multocida isolates were obtained; all of these were of capsular type A apart from one quail isolate of type D. The climate changes, breed, age, location, clinical symptoms, and sample type had impacts on the incidence rate of P. multocida in the hosts.Breed was the most significant factor in chickens, while age and breed were less important in other species (Tables 4  and 5).
In chicken and ducks, the most susceptible breed was Baladi with an average age of 8-9 weeks; diseased samples recorded a higher prevalence than dead birds.
In quails, age as the most affectable factor with a range of 4-5 weeks.In turkeys, the type of sample and age were the influential factors; the higher prevalence was recorded in dead samples with an average age of 9 weeks.
All quail isolates showed absolute resistance to amoxicillin-clavulanic acid, ampicillin, colistin, sulfamethoxazole-trimethoprim, and erythromycin.However, all turkey isolates were susceptible to ampicillin and sulfamethoxazole-trimethoprim.The average MAR index of all P. multocida was 0.43, ranging from 0.23 to 0.77.The highest MAR index (0.77) was found in an isolate recovered from a duck which represent XDR pattern of resistance (Table 6).
Florfenicol and enrofloxacin were the drugs of choice for all the obtained avian isolates.
The association of various isolates is shown in Fig. 4. The analyses of antimicrobial resistance and virulence features revealed that none of the isolates were identical in their profile.They fell into two big clusters; a bigger one with 19 isolates and a smaller one with 11 isolates.Each of these clusters was composed of isolates from different animals and breeds.The small cluster contained isolates only from bird organs, whereas the big cluster was formed from isolates from both organs and tracheal swabs.There was no particular clustering of analyzed genes or phenotypes.Moreover, the heatmap shows that Baladi chickens are more likely to have resistance to multiple antimicrobials, including ampicillin, amoxicillin-clavulanic acid, cephradine, doxycycline, erythromycin, colistin, and sulfamethoxazole-trimethoprim.These isolates are also more likely to have virulence genes, such as those that encode for adhesins, protections, and enzymes that help the bacteria to evade the host's immune system.
As shown in Fig. 5 and Table S1, we identified a high positive significant correlation between lincomycin and colistin-resistant phenotypes (R = 1, p-value < 0.0001) as well as a significant moderate positive correlation between colistin and erythromycin (R = 0.6, p-value = 0.001).On the other side, neomycin and sulfamethoxazoletrimethoprim correlated significantly and negatively (R = − 0.8, p-value < 0.0001).Some antimicrobial resistance genes correlated moderately and significantly negatively (for instance tetH and ermX; R = − 0.6, p-value = 0.0006).Overall, a general negative correlation between antimicrobial resistance and virulence genes was observed with both dfrA1 and pmHAS correlating moderately negatively (R = − 0.5, p-value = 0.007).The p-values in Table S1 show that the correlations are statistically significant meaning that the correlations are not due to chance and are likely to be real.
The graph in Fig. 6 shows that the frequency of occurrences of sodA, ompH, ermX, and blaROB-1 genes are the most prevalent, especially in quails compared to other avian species.Also, Table 7 shows that the frequency of antimicrobial resistance and virulence genes is higher in ducks than in chickens, quails, and turkeys.This suggests that ducks are more likely to be exposed to antimicrobial-resistant and virulent bacteria than other species.
The oma87, sodA, and ptfA virulence genes were found in all (100%) examined avian species.However, sodC was detected in all isolates recovered from ducks.Furthermore, sodC and hgbA were found in turkeys and chickens with percentages of 100 and 80%, respectively.The nanH and toxA genes were not exhibited in any turkey isolates.
The map in Fig. 7 shows that the highest number of cases of pasteurellosis is in the Fakous district followed by the Abo kbir, Belbis, and Dereb negm districts.

Discussion
Pasteurellosis has long been regarded as a serious economic burden on birds and other live animals.Due to its Studies of pathognomonic features of P. multocida isolates among various avian species have shown significant genetic variations among P. multocida strains from each avian species (Hurtado et al. 2020).These variations were attributed to many factors, including difference in host immune systems, various environmental conditions, and different transmission routes which have implications for the pathogenicity, diagnosis, treatment, and prevention of P. multocida infections in avian species (Saha et al. 2021;Shalaby et al. 2021).
Accompanying respiratory signs can reduce the feed intake leading to weight loss with the rapid progression of the disease.Congested catarrhal tracheitis with yellowish exudates is in harmony with those obtained by Marien (2007).Edematous firm yielded blood-cut surface-tinged exudate and mild hemorrhage was detected in P. multocida infection in complete accordance with those mentioned by Lakshman et al. (2006).
The tracheal lesions exhibited no characteristic lesion specified by the isolated strains in all examined birds.Detected lesions in ducks were more severe than those observed in chicken, quails, and turkeys which was similar to that reported by Awadin et al. (2017), however, we disagree with their findings regarding multiple granulomata in P. multocida infection.
Acute interstitial pneumonia was the most prominent lesion observed in acute respiratory infection of the lungs closely related to that described by Lakshman et al. (2006) who described the same lesion characterized by thickening of interalveolar septa with fibrin threads accumulation.Hemorrhages, congestion, and pneumonic changes were in line with the findings of Cynthia and Kahn (2005) and Ram and Abraham (2013) in the case of avian pasteurellosis.
In this study, P. multocida was isolated with a total prevalence of 9.4% (30/317) from the tested birds.The majority were of capsular type A (96.6%; 29/30).This is consistent with other studies (Mohamed and Mageed 2014;Abd-Elsadek et al. 2021;Shalaby et al. 2021) which isolated P. multocida in nearly similar percentages and found capsular type A to be the most common capsular type among avian strains.Interestingly, capsular type D was detected in 3.4% (1/30) of isolated P. multocida from quails, a serogroup considered rare (Glisson et al. 2008), demonstrating a variable prevalence of the serogroups in cases of respiratory disease according to the geographic region (Davies et al. 2003).
Climate change is a major environmental factor that can affect the transmission of this rare serogroup and wide spread of this infectious disease (Lafferty 2009;Bartlow et al. 2019).Pasteurella multocida is more likely to survive and spread in warm, moist conditions (Iverson et al. 2016).As a result, climate change is expected to lead to an increase in the isolation rate of P. multocida isolates among various avian species.
A study published in 2017 found that the isolation rate of P. multocida isolates from poultry flocks in the United States increased by 20% between 1996 and 2016 (Nhung et al. 2017).The study also found that the increase in isolation rate was more pronounced in warmer regions of the country.
Another study, published in 2020 found that the survival of P. multocida in water droplets was significantly longer at temperatures of 37 °C and 42 °C than at 25 °C (Van Driessche et al. 2020).This suggests that climate change could lead to an increase in the transmission of P. multocida through contaminated water.The impact of climate change on the isolation rate of P. multocida isolates is likely to vary depending on the specific region and the species of bird, however, the overall trend is expected to be an increase in isolation rate, as warmer temperatures and more extreme weather events create more favorable conditions for the bacterium to survive and spread (Wilson and Ho 2013).
In addition to climate change, other factors that can affect the isolation rate of P. multocida isolates include the density of bird populations, the presence of stress factors, such as  overcrowding or poor nutrition, and the vaccination status of the birds (Iverson 2015).
The study also found that the breed, age, location, clinical symptoms, and sample type all had a substantial impact on the incidence rate of P. multocida in all hosts suggesting that several factors can contribute to the transmission and spread of P. multocida infections in avian species and give obvious explanations of the obtained prevalence rates.
It is well known that the toxA gene, which encodes the PMT-P.multocida toxin, is substantially connected to serogroup D. This toxin causes osteolysis, a crucial phase that follows the respiratory syndrome (Davies et al. 2003).
The primary requirement for bacterial infection is their attachment to the host cell, thus adhesion is regarded as one of the potential virulence factors (Sarangi et al. 2014 andEssawi et al. 2020).The high prevalence of several adhesion-related genes, including fimA, pfhA, and ptfA, was found in this study, indicating that either proteins operate synergistically or are necessary at various phases of colonization or infection.Filamentous hemagglutinins encoded by the pfhA gene play an important role in the initial colonization of the upper respiratory tract and the frequency of this gene varies greatly among strains of P. multocida.It has been established that this gene is a significant epidemiological marker and its prevalence among 50% of the obtained isolates is connected to the development of disease in several avian species (Haghnazari et al. 2017;Li et al. 2018).Interestingly, the absolute incidence of ptfA was recorded at 100% irrespective of its capsular type.These data were in the line with several prior studies (Sarangi et al. 2015;Furian et al. 2016;Vu-Khac et al. 2020).
F u r t h e r m o r e , t h e p o r i n g e n e s o m p A , ompH, and oma87 were found in 46.6%, 76.6%, and 100% of the isolates, respectively.These results imply that OMPs play a major role in host-pathogen interaction (He et al. 2021).
Diverse virulence profiles were detected for each isolate in correlation with their host or species, indicating differences in the P. multocida strains' capacity for invasion (Prajapati et al. 2020;El Damaty et al. 2023).
High frequencies of resistant genes were noticed towards erythromycin, β-lactam, and colistin in all species of examined birds.These results are not surprising as the isolates showed high resistance to erythromycin, β-lactam, and colistin antimicrobial drugs in susceptibility assays, which is comparable with prior findings in Egypt (Shalaby et al. 2021), Brazil (de Alcântara et al. 2020) and India (Sivagami et al. 2020), but our results were more sever and higher in rates.Additionally, the dfrA1 gene which represents a resistance pattern to trimethoprim was detected at a higher rate in quail species.
Presence of these genes on plasmids poses a great hazard to public health as bacteria can acquire a single plasmid to become multi-antibiotic resistant all at once, eventually developing multidrug resistance.Furthermore, plasmids frequently contain genes that affect the pathogenicity of bacteria (Cao et al. 2020), which explains the obtained high values of multiple resistance indices among various isolates and the appearance of extreme drug resistance (XDR) phenomenon.
The XDR of P. multocida in poultry is a serious threat to animal and public health as it is so difficult to treat infected birds and can lead to high mortality rates (Anholt et al. 2017;Algammal et al. 2022b).
Furthermore, it can be transmitted to humans, either through contact with infected animals or through the consumption of contaminated food posing a serious health risk, as it can cause severe infections, such as pneumonia and meningitis (Phillips et al. 2004).
There are a number of factors that can contribute to the emergence of P. multocida in poultry including the overuse and misuse of antibiotics, the inadequate biosecurity measures, and the transportation and movement of birds between farms (Elayaraja et al. 2020;Bester et al. 2022).
The data have provided valuable insights into the association of antimicrobial resistance and virulence features in P. multocida isolates from various avian species.No particular clustering of analyzed genes or phenotypes was observed suggesting that the presence of antimicrobial resistance and virulence genes is not necessarily correlated.However, the study did find that there was a high positive significant correlation between lincomycin and colistin-resistant phenotypes.This suggests that the presence of one of these resistance genes may increase the likelihood of the presence of the other and a broad-spectrum approach to antimicrobial treatment may be necessary.
The study found that the frequency of occurrences of sodA, ompH, ermX, and blaROB-1 genes is the most prevalent, especially in quails higher than in other understudied.This is attributed to many factors, such as the high density of these birds in commercial poultry farms, the use of antimicrobials in poultry farming, and poor hygiene practices used in the handling of birds.
In total, this study not only helped to represent a complete picture of the diagnosis of pasteurellosis but also aided in understanding the epidemiology of P. multocida infections among avian species.
Notably, the high number of cases in the Fakous district is likely due to several factors, including; the high density of livestock in the district, poor hygiene practices used in the handling of livestock, lack of access to clean water and sanitation and recent climate changes.
These findings highlight the need for interventions to improve hygiene practices used in livestock handling and to improve access to clean water and sanitation in the Fakous Colors indicate number of positive cases per district; the more intense the color, the higher the occurrence district, which could help to reduce the number of cases of pasteurellosis in the district.
Moreover, the obtained different pathogenic and genetic profiles of the isolates can help in the development of molecular diagnostic tests and vaccines for P. multocida infections in the poultry industry.

Conclusion
The present study provides comprehensive epidemiological information on the diversity, histopathological, and virulence gene properties of Egyptian Pasteurella multocida isolates among various avian species.Moreover, the obtained data highlights the growing threat of factors that affect the isolation rate of P. multocida and antimicrobial resistance in examined bird species.The increasing prevalence of antimicrobial resistance and virulence genes in P. multocida observed during this study is a serious public health concern.Therefore, this research is essential to develop effective strategies to prevent the spread and control avian cholera, other diseases, and antimicrobial resistance caused by this bacterium, thus contributing to the great protection of human health.

Fig. 1
Fig. 1 Photomicrograph of H&E-stained sections of P. multocida infected tracheas in different examined birds revealed A: Cystic dilation of some mucosal glands (arrowhead) in quail.B: Detached cilia with haemorrhage (arrow) and cellular infiltration (arrowhead) in Chicken.C: Destructed mucosa with severe extravasated erythrocytes (haemorrhage) (arrowhead) in Turkey.D: Necrosis of some chondrocytes of tracheal cartilage (arrowhead) in Duck.E: Complete destruction of mucosa and tracheal glands (arrowhead) with normal cartilage in Duck.F: Partial destruction of mucosa and submu-

Fig. 2 Fig. 3
Fig. 2 Photomicrograph of H&E-stained sections of P. multocida infected lungs in different examined birds revealed A: Pneumonia represented in diffuse congestion (arrows) and cellular infiltration (arrowhead) in Turkey.B: Severe focal hemorrhage (arrows) with focal cellular infiltration (arrowhead) in Duck.C: Perivascular cellular infiltration (arrow) with focal partial alveolar stenosis (arrowhead) in Quail.D: Perivascular oedema, fibrosis, and cellular

Fig. 4 Fig. 5
Fig. 4 Heatmap supported by dendrogram showing the overview of the distribution of antimicrobial resistance phenotype, genes and virulence genes in the studied bird species.Different bird species,

Fig. 7
Fig. 7 Epidemiological mapping of positive cases of Pasteurellosis in different districts in Sharkia Governorate, Egypt.Colors indicate number of positive cases per district; the more intense the color, the higher the occurrence Abbas AM, Abd El-Moaty DAM,Zaki ESA et al (2018)  Use of molecular biology tools for rapid identification and characterization of Pasteurella spp.Vet World 11:1006 Abd El-Hamid MI, El-Sayed ME, Ali AR et al (2019) Marjoram extract down-regulates the expression of Pasteurella multocida adhesion, colonization and toxin genes: a potential mechanism for its antimicrobial activity.Comp Immunol Microbiol Infect Dis 62:101-108 Abd-Elsadek E, Mostafa AEH, Abouelkhair A (2021) Molecular studies on Pasteurella multocida in ducks.J Curr Vet Res 3:1-9 Abdolmaleki Z, Mashak Z, Safarpoor Dehkordi F (2019) Phenotypic and genotypic characterization of antibiotic resistance in the methicillin-resistant Staphylococcus aureus strains isolated from hospital cockroaches.Antimicrob Resist Infect Control 8:1-14 Algammal AM, Hashem HR, Al-Otaibi AS et al (2021) Emerging MDR-Mycobacterium avium subsp.avium in house-reared domestic birds as the first report in Egypt.BMC Microbiol 21:1-11 Algammal AM, Abo Hashem ME, Alfifi KJ et al (2022a) Sequence analysis, antibiogram profile, virulence and antibiotic resistance genes of XDR and MDR Gallibacterium anatis isolated from layer chickens in Egypt.Infect Drug Resist 15:4321-4334 Algammal AM, Ibrahim RA, Alfifi KJ et al (2022b) A first report of molecular typing, virulence traits, and phenotypic and genotypic resistance patterns of newly emerging XDR and MDR Aeromonas veronii in Mugil seheli.Pathogens 11:1262 Algammal AM, Eidaroos NH, Alfifi KJ et al (2023) oprL gene sequencing, resistance patterns, virulence genes, quorum sensing and antibiotic resistance genes of XDR Pseudomonas aeruginosa isolated from broiler chickens.Infect Drug Resist 16:853-867 Anholt RM, Klima C, Allan N et al (2017) Antimicrobial susceptibility of bacteria that cause bovine respiratory disease complex in Alberta.Canada Front Vet Sci 4:207 Apinda N, Nambooppha B, Rittipornlertrak A et al (2020) Protection against fowl cholera in ducks immunized with a combination vaccine containing live attenuated duck enteritis virus and recombinant outer membrane protein H of Pasteurella multocida.Avian Pathol 49:221-229 Aski HS, Tabatabaei M (2016) Occurrence of virulence-associated genes in Pasteurella multocida isolates obtained from different hosts.Microb Pathog 96:52-57 Awadin W, Ghaly S, Elsawak A (2017) Pathological and immunohistochemical study of P. Multocida capsular type a in tissues of chickens and ducks infected with fowl cholera.Assiut Vet Med J 63:1-18 Bartlow AW, Manore C, Xu C et al (2019) Forecasting zoonotic infectious disease response to climate change: mosquito vectors and a changing environment.Vet Sci 6:40 Bauer AW, Kirby WM, Sherris JC, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method.Am J Clin Pathol 45:493 Bester C, Marschik T, Schmoll F, Käsbohrer A (2022) Development of an economic model to assess the cost-effectiveness of biosecurity measures to reduce the burden of Salmonella and Hepatitis E virus in the pork production chain.In: 20th Congress of the International Society for Animal Hygiene.p 107 Cao Y-P, Lin Q-Q, He W-Y et al (2020) Co-selection may explain the unexpectedly high prevalence of plasmid-mediated colistin resistance gene mcr-1 in a Chinese broiler farm.Zool Res 41:569

Table 1
Oligonucleotide primer sequences used in this study

Table 2
Lesions score of respiratory disorders in trachea of infected birds

Table 3
Lesions score of respiratory disorders in lung of infected birds

Table 6
Source, capsular type, antimicrobial resistance profiles, virulence, and resistance genes of P. multocida isolates recovered from different examined avian species AMC amoxicillin-clavulanic acid, AMP ampicillin, CE cephradine; DO doxycycline; FFC florfenicol; SXT sulfamethoxazole-trimethoprim; CT colistin, E erythromycin; L lincomycin; SH spectinomycin and ENR enrofloxacin a The isolates were resistant to ≥ 1 agent in ≥ 3 antimicrobial categories b The isolates were resistant to ≥ 1 agent in all except ≤ 2 antimicrobial categories c Multiple antibiotic resistance index (average MAR index = 0.43)

Table 7
Frequency of antimicrobial resistance phenotypes, genotypes and virulence genes in the studied birds AK amikacin; N neomycin; AMC amoxicillin-clavulanic acid, AMP ampicillin, CE cephradine; DO doxycycline; FFC florfenicol; SXT sulfamethoxazole-trimethoprim; CT colistin, E erythromycin; L lincomycin; SH spectinomycin and ENR enrofloxacin *p-values refer to significance of differences among studied animal species using Chi-square test.p-value cutoff was 0.05.