Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Clinical and Molecular Phylogenetic Detection of Trueperella pyogenes in Abscessed-wounds of Cattle

T
Tamadhir A. Al-Hamed1
0000-0002-6294-3683
H
Hawraa F.H. Al-Abedi2
0000-0003-1693-0424
N
Noor K.H. Al-Quraishy3
0009-0000-3143-7852
I
Israa M. Essa4
0000-0002-7379-7503
H
Hasanain A.J. Gharban5,*
0000-0002-6438-1450
1Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Basrah, Basra, Iraq.
2Department of Biology, Collage of Education for Pure Sciences, University of Al-Hamdaniya, Al- Hamdaniya, Nineveh, Iraq.
3Department of Anatomy and Histology, College of Veterinary Medicine, University of Wasit, Wasit, Iraq.
4Department of Public Health, College of Veterinary Medicine, University of Basrah, Basra, Iraq.
5Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit, Wasit, Iraq.

ABSTRACT

Background: Abscessed wounds in cattle represent a significant veterinary concern, often leading considerable economic losses due to decreased productivity, treatment cost, potential culling and even death. This study aims to investigate the incidence rate of Trueperella pyogenes in abscessed wounds of cattle using molecular technique with phylogenetic analysis of study isolates and estimation the relationship of infection to animal risk factors.

Methods: A total of 271 cattle of different ages and sexes admitted to private veterinarian clinics or being visited to their farms in Wasit province (Iraq) were diagnosed clinically with abscessed wounds. Then, samples of abscess swab were collected and examined molecularly by the conventional PCR. Finally, the positive T. pyogenes isolates were sequenced, submitted in the NCBI database and analysed phylogenetically. 

Result: Findings of clinical examination revealed a significant increase in abscessed wounds at abdomen but decreased in neck and back when compared to other body regions; head, forelimbs, hindlimbs and pelvis. Targeting of 16S rRNA gene, 7.38% from abscessed wounds of study cattle were positively infected with T. pyogenes. Phylogenetic analysis of the study T. pyogenes isolates revealed its identity to NCBI-BLAST USA T. pyogenes isolate (KX592206.1) at rates of similarity and mutation/changes ranged 98.47-99.86% and 0.0003-0.001%, respectively. Relation to risk factors, incidence and risk of positivity in body parts were elevated significantly in abdomen but decreased in neck and back, head, forelimbs and hindlimbs when compared to pelvis. Concerning age, although insignificant variation in incidence of positivity was seen among age groups of study animals, cattle aged 1-4 years and >4 years were appeared at higher risk of infection than those of <1 year. For sex, also insignificant differences in incidence rate of positivity was detected between females and males; however, females were detected at higher risk of infection than males.

INTRODUCTION

The complexity of wound healing in cattle as in other terrestrial vertebrates involves intricate biological processes influenced by both animal-related factors in addition to nutrition and husbandry practices which play a critical role in facilitating optimal wound healing in cattle; while conversely, disruptions and infections can impair and complicate it (Lux, 2022; Singh et al., 2024). Bacterial pathogens represent the main cause of wound infections which result mostly in various clinical forms that ranged from localized abscesses and folliculitis to more diffuse cellulitis, each presenting unique challenges in diagnosis and management following the prevalence and specific types of bacteria responsible for these purulent conditions that varied geographically and depending on the farm-specific hygiene practices (Malone and Schultz, 2022; Mohammad et al., 2022; Al-Eodawee et al., 2024). Trueperella pyogenes consider one of the most neglected opportunistic pathogens which found usually in mucous membranes and skin of various animals including cattle, pigs and other livestock (Rzewuska et al., 2019). This bacterium can lead to purulent infections in lung, udder, genital tract, liver and skin such as pneumonia, mastitis, metritis as well as liver and skin abscesses (Nagaraja, 2022). Abscesses caused by T. pyogenes can vary in size and location, to resulting sometimes in severe complications like fistulas and arthritis (Rzewuska et al., 2019).
       
Trueperella pyogenes was discovered and classified generally as Bacillus pyogenes in 1893, then to Arcanobacterium pyogenes and recently renamed in honor of the German microbiologist, Hans Georg Trüper. Today, T. pyogenes is classified in the genus of Trueperella that belongs to the family Actinomycetaceae in the order Actinomycetales of the class Actinobacteria (Ashur et al., 2019; Menon, 2024). The bacterium is a Gram-positive, non-motile, non-sporulating, non-capsulated and short rod-shaped facultative anaerobic organism which grown on blood agar as very small, white, opaque and glistening colonies surrounded by a zone of b-hemolysis (Carr, 2017; Saad et al., 2023). Due to limitations of culture methods (fastidious nature, long incubation time due to slow growing, potentially mixed infections and difficulties in identifying), molecular techniques like PCR (polymerase chain reaction) are often used for the detection and identification of T. pyogenes as a rapid, reliable, highly sensitive and specific tool (Mobed et al., 2019; Al-Graibawi et al., 2021; Stefañska et al., 2022; Ehmud et al., 2025). Sequencing methods provide additional advantages in accurate identification, characterization and even detection of antimicrobial resistance by determining specific genes (Boolchandani et al., 2019; Bahloul et al., 2024; Magossi et al., 2025).
               
In Iraq, although T. pyogenes were isolated from the synovial fluid obtained from the knee joint of sheep diagnosed clinically with suppurative arthritis in Mosul (Arslan et al., 2009), milk samples of subclinical mastitic she-camels in middle Euphrates (AL-Tofaily and Al-Roddan, 2011), as well as urine from the urinary balder samples of slaughtered buffaloes (Al-Iraqi et al., 2016) and slaughtered goats (Sadoon, 2021) in Mosul abattoirs. Hence, this study aims to investigate the incidence of T. pyogenes in abscessed wounds of cattle molecularly using conventional PCR assay, sequencing and phylogenetic analysis of study T. pyogenes isolates to determine its identity to the global NCBI-BLAST isolates/strains and estimate the association of infection to animal risk factors including body site of wound, age and sex. 

MATERIALS AND METHODS

Samples
 
This study was conducted during September (2024) to May (2025) on totally 271 cattle of different ages and sexes which attended to the private veterinarian clinics or visited at their farms in Wasit province (Iraq) with abscessed wounds in their bodies. Swab samples were collected from each abscessed wounds of each study animal using the rayon-budded swabs into the ready-to-use-PrimeStore MTM transport tubes (EKF, Germany). All tubes were transported to the Clinical Pathology Lab. (College of Veterinary Medicine/University of Wasit) under cooled conditions and kept frozen until be used for molecular examination. Animal risk factors (body site of wound, age and sex) were reported, also (Almaliky et al., 2024; Jiheel et al., 2025).
 
Molecular examination
 
After preparation of swab samples at room temperature, manufacturer instructions of the PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) were followed. After extraction, all samples of DNAs were checked for purity and concentration by the Nanodrop system (Thermo-scientific, UK) and served for preparation the MasterMix tubes at a final volume 25 μl using the AccuPower® PCR PreMix Kit (Bioneer, Korea) and the designated primer for this study (F: 5´-TTT TGG ATG GGG ATG GGC TC-3´ and R: 5´-TGG CAC ATC GCA GTG TAT GT-3´) based on the NCBI-GenBank T. pyogenes strain (ID: KT191136.1). In the Thermal Cycler system (Bio Rad, USA), the MasterMix tubes were subjected to the conditions of the DNAs amplification as following: 1 cycle for initial denaturation (95°C/7 min), 35 cycles for denaturation (95°C/30 sec), annealing (54°C/30 sec) and extension (72°C/45 sec) and 1 cycle for final extension (72°C/7 min). Electrophoresis for Agarose-gel (1.5%) stained with ethidium bromide was done at 100Volts and 80Am for 90 min and the bands size of positive PCR products was visualized under the UV transilluminator (Clinx Science, China) at an approximately H” 782bp.
 
Sequencing and phylogenetic analysis
 
The positive T. pyogenes isolates were sequenced at the Macrogen Company (South Korea) and the received data were submitted in the NCBI-GenBank to get specific access numbers. Then, ClustralW alignment, multiple sequence alignment and phylogenetic tree analysis was performed using the MEGA-11 Software. 
 
Statistical analysis
 
One-Way ANOVA and t-test in the GraphPad Prism Software version 8.0.2 in addition to the odds ratio (OR) and relative risks (RR) in the MedCalc Statistical Software were applied to detect significant differences between the obtained values at p<0.05 and 95% confidence interval (95%CI), (Al-Abedi et al., 2018).

RESULTS AND DISCUSSION

Clinical examination
 
The findings of clinical examination revealed that the abscessed wounds were increased significantly (p<0.008; 95%CI: 5.116 to 23.46) in abdomen [34.69% (94/271)] but decreased significantly (p<0.05) in neck [4.06% (11/271)] and back [8.49% (23/271)] when compared to other body regions; head [14.02% (38/271)], forelimbs [11.07% (30/271)], hind-limbs [10.33% (28/271)] and pelvis [17.34% (47/271)], (Fig 1). In cattle, skin wounds occur most commonly due to various physical injuries including trauma from fencing, kicks, or falls as well as injuries from other animals or sharp aspects in their environments and can have complications and infections (Wheeler, 2019; Valkova et al., 2021; Faccin et al., 2023). In female, improper milking techniques and mastitis can lead to teat damage and subsequent viral and bacterial infections (Abd-El-Hady, 2015; Sharun et al., 2021); while in males especially adults, warts as the most etiology and other causes (penile hematomas, hair rings and viral infections) can lead to penile lesions and ulcers (Paul, 2024).

Fig 1: Incidence of abscessed wounds in various bodily parts of study animals.


 
Molecular phylogeny
 
Targeting the 16S rRNA gene, 7.38% (20/271) from abscessed wounds of study cattle were positively infected with T. pyogenes (Fig 2). The sequencing data of 20 positive T. pyogenes isolates were submitted in the NCBI database (Cattle-Hass1-Cattle-Hass20). Phylogenetic analysis of the study T. pyogenes isolates revealed its significant identity to the NCBI-BLAST USA T. pyogenes isolate (KX592206.1) at a similarity ranged 98.47-99.86% and mutation/changes ranged 0.0003-0.001% (Fig 3-5, Table 1). In comparison to other studies, Ertaş et al. (2005) investigated the presence of T. pyogenes in abscessed kidney samples of 500 cattle and found that 40% of study samples were positive by PCR. In a study conducted by Liu et al. (2009), 23.5% of endometritic cows were shown a positive reactivity by the PCR assay. Petit et al. (2009) isolated T. pyogenes from the genital tract of 41.3% cows with and without abortion; while, Santos et al. (2010) isolated T. pyogenes from the 86.1% of uterine fluid of Holstein dairy cows. Zastempowska and Lassa (2012) detect the presence of 89 T. pyogenes isolates from the inflamed secretions of 89 dairy cows with clinical mastitis. Ishiyama et al. (2017) detected that the occurrence of T. pyogenes in mastitis cases of 81 Holstein cows was 16.61%. Rezanejad et al. (2019) tested 226 bovine mastitic milk and 172 uterine swabs; in which, positive T. pyogenes isolates were detected in 14.15% and 23.83% samples, respectively. Saad et al. (2023) found that the occurrence of T. pyogenes in raw milk samples of dairy farms was 60% in both cows and buffaloes and 63.33% in milk of farmer houses. Tamai et al. (2023) isolated 65 T. pyogenes strains (453%) from 150 postpartum cattle with clinical abscess symptoms on 22 farms around Tehran (Iran). Wente et al. (2024) detected a total of 151 T. pyogenes isolates among 16 herds with an overall 41 out of 124 isolates (25%) were delivered by one herd. In a recent study, T. pyogenes was isolated from multiple abscesses in spleen of cattle suggesting its role in infections of visceral organs (Hamedi et al., 2025).

Fig 2: Agarose-gel electrophoresis of some positive PCR products to T. pyogenes isolates.



Fig 3: Multiple sequence alignment using the MEGA-11 software.



Fig 4: Multiple sequence alignment using the NCBI-MSA viewer.



Fig 5: Phylogenetic tree analysis of study and NCBI-BLAST T. pyogenes isolates/strains.



Table 1: Homology Sequence identity for local and NCBI-BLAST T. pyogenes isolates.


       
Targeting of 16S rRNA gene, phylogenetic analysis revealed that the study T. pyogenes isolates are close-related to the NCBI-BLAST USA T. pyogenes isolate (KX592206.1) which conducted by Rogovskyy et al. (2018) and recovered from 35 ruminants including 25 cattle, 8 goats and 2 sheep. These data indicate that the local T. pyogenes isolates might geographically widespread, potentially transmitted pathways and having similar virulence factors. Thus, the 16S rRNA gene is a powerful tool for bacterial detection and identification of genetic diversity and can enable researchers to identify T. pyogenes in various samples and environments (Gharban and Yousif, 2021; Kwiecieñ et al., 2024; Saifudeen et al., 2024).
 
Risk factors
 
In the current study, the distribution of positive T. pyogenes infections were varied significantly (p<0.05) among the groups of each factor (Table 2). For body part, incidence of positive T. pyogenes infections as well as values of OR and RR were elevated significantly (p<0.0487, p<0.0001 and p<0.0001, respectively) in abdomen (13.83%, 3.902 and 3.485, respectively) but decreased significantly in neck and back (0%, 0 and 0, respectively), head (2.63%, 0.329 and 0.317, respectively), forelimbs (3.33%, 0.4 and 0.418, respectively) and hindlimbs (3.57%, 0.435 and 0.462, respectively) when compared to pelvis (8.51%, 1.208, 1.197, respectively). Concerning age, although insignificant variation (p<0.0755) was seen in incidence of positive T. pyogenes infections among age groups of study animals, cattle aged 1-4 years and >4 years were reported a significant (p<0.0001) higher values of OR (1.399 and 1.397, respectively) and RR (1.367 and 1.348, respectively) than those of <1 year (0.57 and 0.6). Regarding sex, insignificant differences (p<0.0562) was detected between the incidence rate of positive T. pyogenes infections among females (8.81%) and males (3.85%); however, significant (p<0.0001) higher risk of infection (OR and RR) was observed in females (2.415 and 2.316, respectively) than males (0.414 and 0.432, respectively). Although, there was no clear explanation for this case, we thought that there were problems with inappropriate environments or handling methods, high stocking densities and tying the animal too tightly (Alam et al., 2010). In addition, the increasing incidence of mastitis in adult cows and warts in both males and females might play a role in increasing the risk and occurrence of abscessed wounds (Gharban et al., 2023).

Table 2: Distribution of positive T. pyogenes among groups of animals’ risk factors.


       
Although no significant association was reported between the incidence of T. pyogenes age of study animals, there was a significant elevation in risk of infection in cattle aged 1-4 years and >4 years more than those of <1 years. The relationship between age and infection risk can be complex; however with advancing age, increased exposure to several pathogens and waning of immunity might explain our results (Vlasova and Saif, 2021). Our results showed that female cattle were at a higher risk of T. pyogenes infection than males. This might be attributed to anatomical and physiological differences since skin of males is thicker particularly in dermis and may have higher density of collagen and sebaceous glands than females (Yang et al., 2017; Tarique et al., 2021). Also, female cattle experienced more skin wounds due to mastitis that leading to increase susceptibility and skin damage; as well as during pregnancy that increase pressure on abdomen from the growing fetus (Zhao and Lacasse, 2008; More et al., 2017).

CONCLUSION

This might represent the first Iraqi study indicates the incidence of T. pyogenes in cattle suggesting the need to furthermore studies in other bovine infections as well as in other animals’ infection. Molecular phylogeny demonstrates a high reliability in detection of the bacterium in the swabs of abscess wounds; therefore, utilization of molecular techniques can support the cost, time and effort in detection of such infections. However, effective wound management necessitates a comprehensive evaluation of the wound itself, coupled with a systemic examination of the animal’s overall health and environmental condition. Also, successful treatment could be obtained by regular draining of pus along with daily antiseptic dressing and parenteral administration of antibiotics.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    Full Research Article

    Clinical and Molecular Phylogenetic Detection of Trueperella pyogenes in Abscessed-wounds of Cattle

    T
    Tamadhir A. Al-Hamed1
    0000-0002-6294-3683
    H
    Hawraa F.H. Al-Abedi2
    0000-0003-1693-0424
    N
    Noor K.H. Al-Quraishy3
    0009-0000-3143-7852
    I
    Israa M. Essa4
    0000-0002-7379-7503
    H
    Hasanain A.J. Gharban5,*
    0000-0002-6438-1450
    1Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Basrah, Basra, Iraq.
    2Department of Biology, Collage of Education for Pure Sciences, University of Al-Hamdaniya, Al- Hamdaniya, Nineveh, Iraq.
    3Department of Anatomy and Histology, College of Veterinary Medicine, University of Wasit, Wasit, Iraq.
    4Department of Public Health, College of Veterinary Medicine, University of Basrah, Basra, Iraq.
    5Department of Internal and Preventive Veterinary Medicine, College of Veterinary Medicine, University of Wasit, Wasit, Iraq.

    ABSTRACT

    Background: Abscessed wounds in cattle represent a significant veterinary concern, often leading considerable economic losses due to decreased productivity, treatment cost, potential culling and even death. This study aims to investigate the incidence rate of Trueperella pyogenes in abscessed wounds of cattle using molecular technique with phylogenetic analysis of study isolates and estimation the relationship of infection to animal risk factors.

    Methods: A total of 271 cattle of different ages and sexes admitted to private veterinarian clinics or being visited to their farms in Wasit province (Iraq) were diagnosed clinically with abscessed wounds. Then, samples of abscess swab were collected and examined molecularly by the conventional PCR. Finally, the positive T. pyogenes isolates were sequenced, submitted in the NCBI database and analysed phylogenetically. 

    Result: Findings of clinical examination revealed a significant increase in abscessed wounds at abdomen but decreased in neck and back when compared to other body regions; head, forelimbs, hindlimbs and pelvis. Targeting of 16S rRNA gene, 7.38% from abscessed wounds of study cattle were positively infected with T. pyogenes. Phylogenetic analysis of the study T. pyogenes isolates revealed its identity to NCBI-BLAST USA T. pyogenes isolate (KX592206.1) at rates of similarity and mutation/changes ranged 98.47-99.86% and 0.0003-0.001%, respectively. Relation to risk factors, incidence and risk of positivity in body parts were elevated significantly in abdomen but decreased in neck and back, head, forelimbs and hindlimbs when compared to pelvis. Concerning age, although insignificant variation in incidence of positivity was seen among age groups of study animals, cattle aged 1-4 years and >4 years were appeared at higher risk of infection than those of <1 year. For sex, also insignificant differences in incidence rate of positivity was detected between females and males; however, females were detected at higher risk of infection than males.

    INTRODUCTION

    The complexity of wound healing in cattle as in other terrestrial vertebrates involves intricate biological processes influenced by both animal-related factors in addition to nutrition and husbandry practices which play a critical role in facilitating optimal wound healing in cattle; while conversely, disruptions and infections can impair and complicate it (Lux, 2022; Singh et al., 2024). Bacterial pathogens represent the main cause of wound infections which result mostly in various clinical forms that ranged from localized abscesses and folliculitis to more diffuse cellulitis, each presenting unique challenges in diagnosis and management following the prevalence and specific types of bacteria responsible for these purulent conditions that varied geographically and depending on the farm-specific hygiene practices (Malone and Schultz, 2022; Mohammad et al., 2022; Al-Eodawee et al., 2024). Trueperella pyogenes consider one of the most neglected opportunistic pathogens which found usually in mucous membranes and skin of various animals including cattle, pigs and other livestock (Rzewuska et al., 2019). This bacterium can lead to purulent infections in lung, udder, genital tract, liver and skin such as pneumonia, mastitis, metritis as well as liver and skin abscesses (Nagaraja, 2022). Abscesses caused by T. pyogenes can vary in size and location, to resulting sometimes in severe complications like fistulas and arthritis (Rzewuska et al., 2019).
           
    Trueperella pyogenes     was discovered and classified generally as Bacillus pyogenes in 1893, then to Arcanobacterium pyogenes and recently renamed in honor of the German microbiologist, Hans Georg Trüper. Today, T. pyogenes is classified in the genus of Trueperella that belongs to the family Actinomycetaceae in the order Actinomycetales of the class Actinobacteria (Ashur et al., 2019; Menon, 2024). The bacterium is a Gram-positive, non-motile, non-sporulating, non-capsulated and short rod-shaped facultative anaerobic organism which grown on blood agar as very small, white, opaque and glistening colonies surrounded by a zone of b-hemolysis (Carr, 2017; Saad et al., 2023). Due to limitations of culture methods (fastidious nature, long incubation time due to slow growing, potentially mixed infections and difficulties in identifying), molecular techniques like PCR (polymerase chain reaction) are often used for the detection and identification of T. pyogenes as a rapid, reliable, highly sensitive and specific tool (Mobed et al., 2019; Al-Graibawi et al., 2021; Stefañska et al., 2022; Ehmud et al., 2025). Sequencing methods provide additional advantages in accurate identification, characterization and even detection of antimicrobial resistance by determining specific genes (Boolchandani et al., 2019; Bahloul et al., 2024; Magossi et al., 2025).
                   
    In Iraq, although T. pyogenes were isolated from the synovial fluid obtained from the knee joint of sheep diagnosed clinically with suppurative arthritis in Mosul (Arslan et al., 2009), milk samples of subclinical mastitic she-camels in middle Euphrates (AL-Tofaily and Al-Roddan, 2011), as well as urine from the urinary balder samples of slaughtered buffaloes (Al-Iraqi et al., 2016) and slaughtered goats (Sadoon, 2021) in Mosul abattoirs. Hence, this study aims to investigate the incidence of T. pyogenes in abscessed wounds of cattle molecularly using conventional PCR assay, sequencing and phylogenetic analysis of study T. pyogenes isolates to determine its identity to the global NCBI-BLAST isolates/strains and estimate the association of infection to animal risk factors including body site of wound, age and sex. 

    MATERIALS AND METHODS

    Samples
     
    This study was conducted during September (2024) to May (2025) on totally 271 cattle of different ages and sexes which attended to the private veterinarian clinics or visited at their farms in Wasit province (Iraq) with abscessed wounds in their bodies. Swab samples were collected from each abscessed wounds of each study animal using the rayon-budded swabs into the ready-to-use-PrimeStore MTM transport tubes (EKF, Germany). All tubes were transported to the Clinical Pathology Lab. (College of Veterinary Medicine/University of Wasit) under cooled conditions and kept frozen until be used for molecular examination. Animal risk factors (body site of wound, age and sex) were reported, also (Almaliky et al., 2024; Jiheel et al., 2025).
     
    Molecular examination
     
    After preparation of swab samples at room temperature, manufacturer instructions of the PrestoTM Mini gDNA Bacteria Kit (Geneaid, Taiwan) were followed. After extraction, all samples of DNAs were checked for purity and concentration by the Nanodrop system (Thermo-scientific, UK) and served for preparation the MasterMix tubes at a final volume 25 μl using the AccuPower® PCR PreMix Kit (Bioneer, Korea) and the designated primer for this study (F: 5´-TTT TGG ATG GGG ATG GGC TC-3´ and R: 5´-TGG CAC ATC GCA GTG TAT GT-3´) based on the NCBI-GenBank T. pyogenes strain (ID: KT191136.1). In the Thermal Cycler system (Bio Rad, USA), the MasterMix tubes were subjected to the conditions of the DNAs amplification as following: 1 cycle for initial denaturation (95°C/7 min), 35 cycles for denaturation (95°C/30 sec), annealing (54°C/30 sec) and extension (72°C/45 sec) and 1 cycle for final extension (72°C/7 min). Electrophoresis for Agarose-gel (1.5%) stained with ethidium bromide was done at 100Volts and 80Am for 90 min and the bands size of positive PCR products was visualized under the UV transilluminator (Clinx Science, China) at an approximately H” 782bp.
     
    Sequencing and phylogenetic analysis
     
    The positive T. pyogenes isolates were sequenced at the Macrogen Company (South Korea) and the received data were submitted in the NCBI-GenBank to get specific access numbers. Then, ClustralW alignment, multiple sequence alignment and phylogenetic tree analysis was performed using the MEGA-11 Software. 
     
    Statistical analysis
     
    One-Way ANOVA and t-test in the GraphPad Prism Software version 8.0.2 in addition to the odds ratio (OR) and relative risks (RR) in the MedCalc Statistical Software were applied to detect significant differences between the obtained values at p<0.05 and 95% confidence interval (95%CI), (Al-Abedi et al., 2018).

    RESULTS AND DISCUSSION

    Clinical examination
     
    The findings of clinical examination revealed that the abscessed wounds were increased significantly (p<0.008; 95%CI: 5.116 to 23.46) in abdomen [34.69% (94/271)] but decreased significantly (p<0.05) in neck [4.06% (11/271)] and back [8.49% (23/271)] when compared to other body regions; head [14.02% (38/271)], forelimbs [11.07% (30/271)], hind-limbs [10.33% (28/271)] and pelvis [17.34% (47/271)], (Fig 1). In cattle, skin wounds occur most commonly due to various physical injuries including trauma from fencing, kicks, or falls as well as injuries from other animals or sharp aspects in their environments and can have complications and infections (Wheeler, 2019; Valkova et al., 2021; Faccin et al., 2023). In female, improper milking techniques and mastitis can lead to teat damage and subsequent viral and bacterial infections (Abd-El-Hady, 2015; Sharun et al., 2021); while in males especially adults, warts as the most etiology and other causes (penile hematomas, hair rings and viral infections) can lead to penile lesions and ulcers (Paul, 2024).

    Fig 1: Incidence of abscessed wounds in various bodily parts of study animals.


     
    Molecular phylogeny
     
    Targeting the 16S rRNA gene, 7.38% (20/271) from abscessed wounds of study cattle were positively infected with T. pyogenes (Fig 2). The sequencing data of 20 positive T. pyogenes isolates were submitted in the NCBI database (Cattle-Hass1-Cattle-Hass20). Phylogenetic analysis of the study T. pyogenes isolates revealed its significant identity to the NCBI-BLAST USA T. pyogenes isolate (KX592206.1) at a similarity ranged 98.47-99.86% and mutation/changes ranged 0.0003-0.001% (Fig 3-5, Table 1). In comparison to other studies, Ertaş et al. (2005) investigated the presence of T. pyogenes in abscessed kidney samples of 500 cattle and found that 40% of study samples were positive by PCR. In a study conducted by Liu et al. (2009), 23.5% of endometritic cows were shown a positive reactivity by the PCR assay. Petit et al. (2009) isolated T. pyogenes from the genital tract of 41.3% cows with and without abortion; while, Santos et al. (2010) isolated T. pyogenes from the 86.1% of uterine fluid of Holstein dairy cows. Zastempowska and Lassa (2012) detect the presence of 89 T. pyogenes isolates from the inflamed secretions of 89 dairy cows with clinical mastitis. Ishiyama et al. (2017) detected that the occurrence of T. pyogenes in mastitis cases of 81 Holstein cows was 16.61%. Rezanejad et al. (2019) tested 226 bovine mastitic milk and 172 uterine swabs; in which, positive T. pyogenes isolates were detected in 14.15% and 23.83% samples, respectively. Saad et al. (2023) found that the occurrence of T. pyogenes in raw milk samples of dairy farms was 60% in both cows and buffaloes and 63.33% in milk of farmer houses. Tamai et al. (2023) isolated 65 T. pyogenes strains (453%) from 150 postpartum cattle with clinical abscess symptoms on 22 farms around Tehran (Iran). Wente et al. (2024) detected a total of 151 T. pyogenes isolates among 16 herds with an overall 41 out of 124 isolates (25%) were delivered by one herd. In a recent study, T. pyogenes was isolated from multiple abscesses in spleen of cattle suggesting its role in infections of visceral organs (Hamedi et al., 2025).

    Fig 2: Agarose-gel electrophoresis of some positive PCR products to T. pyogenes isolates.



    Fig 3: Multiple sequence alignment using the MEGA-11 software.



    Fig 4: Multiple sequence alignment using the NCBI-MSA viewer.



    Fig 5: Phylogenetic tree analysis of study and NCBI-BLAST T. pyogenes isolates/strains.



    Table 1: Homology Sequence identity for local and NCBI-BLAST T. pyogenes isolates.


           
    Targeting of 16S rRNA gene, phylogenetic analysis revealed that the study T. pyogenes isolates are close-related to the NCBI-BLAST USA T. pyogenes isolate (KX592206.1) which conducted by Rogovskyy et al. (2018) and recovered from 35 ruminants including 25 cattle, 8 goats and 2 sheep. These data indicate that the local T. pyogenes isolates might geographically widespread, potentially transmitted pathways and having similar virulence factors. Thus, the 16S rRNA gene is a powerful tool for bacterial detection and identification of genetic diversity and can enable researchers to identify T. pyogenes in various samples and environments (Gharban and Yousif, 2021; Kwiecieñ et al., 2024; Saifudeen et al., 2024).
     
    Risk factors
     
    In the current study, the distribution of positive T. pyogenes infections were varied significantly (p<0.05) among the groups of each factor (Table 2). For body part, incidence of positive T. pyogenes infections as well as values of OR and RR were elevated significantly (p<0.0487, p<0.0001 and p<0.0001, respectively) in abdomen (13.83%, 3.902 and 3.485, respectively) but decreased significantly in neck and back (0%, 0 and 0, respectively), head (2.63%, 0.329 and 0.317, respectively), forelimbs (3.33%, 0.4 and 0.418, respectively) and hindlimbs (3.57%, 0.435 and 0.462, respectively) when compared to pelvis (8.51%, 1.208, 1.197, respectively). Concerning age, although insignificant variation (p<0.0755) was seen in incidence of positive T. pyogenes infections among age groups of study animals, cattle aged 1-4 years and >4 years were reported a significant (p<0.0001) higher values of OR (1.399 and 1.397, respectively) and RR (1.367 and 1.348, respectively) than those of <1 year (0.57 and 0.6). Regarding sex, insignificant differences (p<0.0562) was detected between the incidence rate of positive T. pyogenes infections among females (8.81%) and males (3.85%); however, significant (p<0.0001) higher risk of infection (OR and RR) was observed in females (2.415 and 2.316, respectively) than males (0.414 and 0.432, respectively). Although, there was no clear explanation for this case, we thought that there were problems with inappropriate environments or handling methods, high stocking densities and tying the animal too tightly (Alam et al., 2010). In addition, the increasing incidence of mastitis in adult cows and warts in both males and females might play a role in increasing the risk and occurrence of abscessed wounds (Gharban et al., 2023).

    Table 2: Distribution of positive T. pyogenes among groups of animals’ risk factors.


           
    Although no significant association was reported between the incidence of T. pyogenes age of study animals, there was a significant elevation in risk of infection in cattle aged 1-4 years and >4 years more than those of <1 years. The relationship between age and infection risk can be complex; however with advancing age, increased exposure to several pathogens and waning of immunity might explain our results (Vlasova and Saif, 2021). Our results showed that female cattle were at a higher risk of T. pyogenes infection than males. This might be attributed to anatomical and physiological differences since skin of males is thicker particularly in dermis and may have higher density of collagen and sebaceous glands than females (Yang et al., 2017; Tarique et al., 2021). Also, female cattle experienced more skin wounds due to mastitis that leading to increase susceptibility and skin damage; as well as during pregnancy that increase pressure on abdomen from the growing fetus (Zhao and Lacasse, 2008; More et al., 2017).

    CONCLUSION

    This might represent the first Iraqi study indicates the incidence of T. pyogenes in cattle suggesting the need to furthermore studies in other bovine infections as well as in other animals’ infection. Molecular phylogeny demonstrates a high reliability in detection of the bacterium in the swabs of abscess wounds; therefore, utilization of molecular techniques can support the cost, time and effort in detection of such infections. However, effective wound management necessitates a comprehensive evaluation of the wound itself, coupled with a systemic examination of the animal’s overall health and environmental condition. Also, successful treatment could be obtained by regular draining of pus along with daily antiseptic dressing and parenteral administration of antibiotics.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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