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Full Research Article

Prevalence of Uterine Infections and Development of a Multiplex PCR Assay for their Detection in Cattle

P
Priyanka Subhashrao Narwade1
P
Priya Ranjan Kumar2,*
P
Pesingi Pavan Kumar3
B
B. Balamurugan1
D
Dayanidhi Jena2
S
Sanjay Kumar Ravi1
R
Rabindra Mohan Mishra4
M
Mayukh Ghosh5
A
Anshuman Kumar6
1Department of Veterinary Gynaecology and Obstetrics, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
2Unit of Teaching Veterinary Clinical Complex, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
3Department of Veterinary Public Health and Epidemiology, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
4Krishi Vigyan Kendra, Chatra-825 401, Jharkhand India.
5Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
6Department of Animal Genetics and Breeding, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.

ABSTRACT

Background: Uterine infections are a leading cause of infertility and economic loss in dairy cattle, often resulting from complex polymicrobial infections. Rapid and accurate detection of the causative pathogens is essential for effective management. This study focuses on assessing the prevalence of uterine infections and developing a multiplex PCR assay for simultaneous detection of key bacterial pathogens in cattle.

Methods: A total of 1,905 cattle from the Vindhyan region were screened for uterine discharge and categorized as clear with tiny flakes, cloudy, mucopurulent, purulent, or haemorrhagic. Sixty uterine samples were aseptically collected for molecular analysis. Conventional PCR was employed to detect Escherichia coli, Fusobacterium necrophorum, Trueperella pyogenes, Clostridium tertium and Histophilus somni. A multiplex PCR (mPCR) assay was subsequently developed for simultaneous detection of E. coli, C. tertium and H. somni and its sensitivity, specificity and accuracy were assessed to evaluate diagnostic performance.

Result: The overall prevalence of uterine infections was 10.44%, consisting of 60.80% subclinical endometritis, 26.63% clinical endometritis and 12.56% metritis. The prevalence rates of E. coli, C. tertium and H. somni were 85%, 92.9% and 73.33%, respectively. Conventional PCR detected E. coli in 54, F. necrophorum in 01, T. pyogenes in 70, C. tertium in 44 and H. somni in 12 of the 60 uterine samples. Using mPCR, 51 samples were positive for E. coli, 44 for C. tertium and 15 for H. somni. The sensitivity, specificity and accuracy of the mPCR were 100%, 66.6% and 95% for E. coli; 100% each for C. tertium; and 80%, 100% and 95% for H. somni, respectively, indicating its high diagnostic efficiency.

INTRODUCTION

Efficient reproductive performance is fundamental to profitable dairy farming, as it directly impacts herd productivity. However, uterine infections remain a major constraint, contributing significantly to infertility, subfertility and economic loss. Uterine infections, broadly categorized as endometritis, metritis and pyometra depending on their severity and clinical presentation (Azawi, 2008), impair both fertility and productivity. Their economic impact is substantial, arising from delayed conception, increased insemination frequency, prolonged calving intervals, reduced milk yield and premature culling (Deka et al., 2021). The losses are especially severe for smallholder farmers where livestock constitute a primary source of income and nutrition.
       
The prevalence of uterine infections in cattle varies considerably, with studies indicating that nearly half of dairy cows experience some degree of uterine infection during the postpartum period (Comlekcioglu et al., 2024). The incidence is notably higher among high-yielding dairy breeds (Várhidi et al., 2024). Subclinical uterine infections, often overlooked during routine herd management, pose a major challenge as they silently impair fertility. Prevalence data, therefore, are critical for designing effective reproductive management programs. Uterine infections in cattle are typically caused by a variety of bacterial pathogens, including Escherichia coli (E. coli),Trueperella pyogenes (T. pyogenes), Fusobacterium necrophorum (F. necrophorum), Prevotella melaninogenica (P. melaninogenica) Staphylococcus aureus (S. aureus), Streptococcus uberis (S. uberis), Bacteroides spp. and Proteus spp. usually as mixed infections (Drillich and Wagener, 2018; Jeon et al., 2018). Clostridium tertium (C. tertium), Histophilus somni (H. somni) and Shigella spp. were less frequently associated with bovine uterine infections in earlier literature, however emerging as potential pathogens of uterine infections. The complex and polymicrobial etiology of uterine infections poses significant challenges for their diagnosis and management. Traditional diagnostic methods, such as clinical examination, vaginal mucus scoring, uterine cytology and bacteriological culture, have long been used in veterinary practice. However, these methods are often constrained by limited sensitivity, delayed results and the inability to clearly distinguish pathogenic organisms from commensals (Barlund et al., 2008).
       
Recent advances in molecular diagnostics have transformed the understanding of bovine uterine infections. Techniques such as PCR, real-time PCR (RT-PCR), multiplex PCR (mPCR) and sequencing-based methods offer enhanced sensitivity, specificity and rapid detection compared to traditional approaches (Aghamiri et al., 2014; Kumar Chetan et al., 2021; Selim et al., 2024; Tahir et al., 2025). These molecular approaches allow for accurate identification of pathogens, detection of mixed infections and characterization of virulence-associated genes, supporting precise diagnosis. Furthermore, PCR-based assays allow direct detection of bacterial DNA from uterine samples, reduces dependence on viable cultures and facilitates large-scale epidemiological surveillance. These advantages highlight the growing importance of molecular diagnostics in the context of bovine uterine infections. Therefore, the present study was designed to investigate the prevalence of uterine infections in cattle in Vindhyan region of Uttar Pradesh, India and to develop and evaluate the utility of molecular diagnostic approaches in their detection.

MATERIALS AND METHODS

Study area and sample collection
 
A total of 1,905 cattle were screened across the Vindhyan region of Uttar Pradesh, of which, 199 were diagnosed with uterine infections. Among the infected cattle, 60 uterine samples were collected aseptically from Mirzapur, Bhadohi, Varanasi and Sonbhadra districts of Vindhyan region. The uterine fluid was immediately transferred into sterile collection vials. The collected samples were initially preserved at 4°C in 20% glycerol stock to maintain microbial viability and subsequently stored at -20°C until further processing.
 
Categorization of samples and case definition
 
The collected samples were categorized based on physical characteristics of the uterine contents, which included clear with tiny flakes, cloudy, mucopurulent, purulent and bloody discharges. Cows exhibiting clear with tiny flakes were categorized as subclinical endometritis. Animals showing cloudy or mucopurulent discharge were categorized as clinical endometritis whereas, cows with purulent or bloody discharge were categorized as metritis.
 
DNA extraction
 
Genomic DNA from the sample collected was extracted using a GSure® Bacterial DNA Isolation Kit (GCC Biotech, India) as per the manufacturer’s protocol.
 
Primers
 
The nucleotide sequence of E. coli fimH gene, F. necrophorum IlkA gene, T. pyogenes plo gene, C. tertium 16S rRNA gene were retrieved from the NCBI GenBank database and the specific pairs of oligonucleotide primers were designed using primer blast of NCBI. The primers for H. somni 16S rRNA gene was taken from published literature. The designed and published primers were subsequently synthesized commercially at Barcode Biosciences Pvt. Ltd. (Bengaluru, India). The details are presented in Table 1.

Table 1: Primers pairs designed to amplify target genes.



Reaction conditions of PCR
 
PCR conditions were optimized for specific and efficient amplification. Each reaction was performed in a 25 µL volume, containing 12.5 µL of 2X PCR master mix, 1 µL each of forward and reverse primers, 3 µL of diluted DNA template and 7.5 µL of nuclease-free water. The amplification protocol included an initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation, primer annealing and extension. Denaturation was optimized at 95°C for 1 minutes. Annealing temperatures were optimized separately for each primer pair: 65°C for 1 min for the fimH gene, 52°C for 45 s for the IlkA gene, 57°C for 30s for plo gene, 55°C for 30 s for the C. tertium 16S rRNA gene and H. somni 16S rRNA gene. Extension was performed at 72°C for 1 min in each cycle, followed by a final extension at 72°C for 10 min. PCR products (10 µL) were resolved on a 1% agarose gel, stained with ethidium bromide and visualized using a gel documentation system.
 
Sequencing of amplicon
 
Representative products were purified and subjected to Sanger sequencing. Chromatograms were analyzed using Chromas (version 2.6.6) software and sequences were compared with GenBank entries using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
 
Reaction conditions of multiplex PCR
 
Three bacteria namely E. coli, C. tertium and H. somni were selected for mPCR and reaction conditions were standardized. Each mPCR reaction was carried out in a 25 µL volume, containing 12.5 µL of 2X PCR master mix, 6 µL of primers (1 µL each of forward and reverse primers for each gene), 3 µL of diluted DNA template and 3.5 µL of nuclease-free water. The amplification protocol consisted of an initial denaturation at 95°C for 10 min, followed by denaturation 95°C for 1 min, annealing at 59°C for 1 min and extension at 72°C for 1 min, repeated for 30 cycles in a thermocycler. The final extension was carried out at 72°C for 10 min to ensure complete synthesis of amplicons. Each mPCR product (10 µL) was subjected to electrophoresis on a 1% agarose gel, stained with ethidium bromide and visualized using a gel documentation system.
 
Ethical approval
 
All procedures for sample collection and laboratory analysis were performed in accordance with the institutional guidelines for animal experimentation. Ethical clearance was obtained from the Institutional Animal Ethics Committee (IAEC), Faculty of Veterinary and Animal Sciences, Banaras Hindu University, under approval number IAEC/BHU/FVAS/2024/173.

RESULTS AND DISCUSSION

Prevalence of uterine infection
 
Out of a total of 1,905 cattle screened across the Vindhyan region of Uttar Pradesh, 199 animals were diagnosed with uterine infections. The diagnosis was based on a combination of reproductive history, clinical signs and the physical characteristics of the uterine discharge. The overall prevalence of uterine infection was found to be 10.44%. The overall and district-wise prevalence of subclinical endometritis, clinical endometritis and metritis is summarized in Table 2, while the prevalence of E. coli, C. tertium and H. somni in the region is presented in Table 4. These findings indicate a considerable burden of uterine infections among cattle in the region, emphasizing the need for improved reproductive health monitoring and timely diagnosis and therapeutic interventions. Reports show substantial variability in the prevalence of uterine infections in cattle across studies and regions, with some studies reporting rates as high as 50% (Sheldon et al., 2008; Comlekcioglu et al., 2024; Mekibib et al., 2024). The prevalence of metritis has been reported to range from 7.2% to 40% (Várhidi et al., 2024; Nguyen et al., 2025), while clinical endometritis ranges from 5% to 35.5% and subclinical endometritis from 11% to 72% (Galvao, 2012; Aghamiri et al., 2014; Sarkar et al., 2016; Kumar Chetan et al., 2021). The prevalence of E. coli in bovine uterine infections has been reported to range from 33.4% to 72.18% (Bicalho et al., 2010; Dutta et al., 2019; Shafique et al., 2021), whereas literature on the prevalence of C. tertium and H. somni is megre. Variations in reported prevalence rates likely reflect differences in diagnostic methods, criteria for classifying uterine infections, postpartum stages, as well as factors such as parity and herd management practices. (Lewis 1997; Aghamiri et al., 2014). Uterine infections are frequently associated with predisposing reproductive disorders such as dystocia, uterine prolapse, abortion and retained fetal membranes (Bell and Roberts, 2007; Singhal et al., 2011; Honparkhe et al., 2025), which compromise uterine defense mechanisms and facilitate bacterial colonization of the endometrium. These conditions collectively contribute to impaired uterine health and subsequent fertility problems in affected animals.

Table 2: Prevalence of uterine infection across the vindhyan region.


 
Development of multiplex PCR assay
 
In order to develop mPCR assay, conventional PCR was first performed to identify the E. coli, C. tertium, F. necrophorum, T. pyogenes and H. somni as a gold standard test. In the present study E. coli (Fig 1; Table 3) emerged as the predominant uterine pathogen, followed by C. tertium (Fig 4; Table 3) across all categories of uterine discharge. In addition, F. necrophorum (Fig 2; Table 3), T. pyogenes (Fig 3; Table 3) and H. somni (Fig 5; Table 3) were also detected, highlighting the complex microbial interactions contributing to uterine pathology. All the amplicon was confirmed by Sanger sequencing except Plo gene of T. pyogenes which may be due to non-specific amplification. E. coli is often regarded as the primary etiological agent of the uterine infection (Sheldon et al., 2002; Williams et al., 2005; Mekibib et al., 2024), gaining access to the uterine lumen during or shortly after parturition. Subsequently, T. pyogenes and F. necrophorum may establish synergistic infections (Sheldon et al., 2008; Haimerl et al., 2018). The identification of C. tertium and H. somni in the current study is noteworthy, as these organisms are less frequently associated with bovine uterine infections in earlier literature (Olson, 1984; Jeon et al., 2015; Saad et al., 2022; Molín et al., 2024) suggesting potential regional variation in pathogen ecology or identification techniques. The observed variation in bacteriological findings across studies may also be attributed to differences in sampling stage, diagnostic criteria, geographical conditions, animal breed and management practices. Collectively, these findings indicate that while E. coli continue to play a dominant role in uterine infections, emerging bacteria such as C. tertium and H. somni may be gaining epidemiological significance in specific herds or regions, warranting further investigation using molecular and metagenomic approaches.

Table 3: Findings of uterine pathogens using PCR across the vindhyan region.



Fig 1: PCR amplification of E. coli (fimH) gene at 604 bp.



Fig 2: PCR amplification of F. necrophorum (IlkA) gene at 406 bp.



Fig 3: PCR amplification of T. pyogenes (plo) gene at 1010 bp.



Fig 4: PCR amplification of C. tertium 16S rRNA gene at 988 bp.



Fig 5: PCR amplification of H. somni 16S rRNA gene at 313 bp.


       
Based on PCR and Sanger sequencing result, a mPCR assay was successfully developed for the simultaneous detection of E. coli, C. tertium and H. somni (Fig 6 and 7; Table 4). The developed mPCR accurately identified all three pathogens in 90% (54/60) of samples, while 10% (6/60) were correctly identified for two of the three target pathogens. The assay showed high sensitivity and accuracy, with moderate specificity for E. coli, whereas C. tertium and H. somni exhibited high sensitivity, specificity and accuracy (Table 4). Traditionally, T. pyogenes, E. coli, F. necrophorum and P. melaninogenica are considered the major uterine pathogens (Sheldon and Dobson, 2004; Azawi, 2008); however, recent studies have highlighted emerging pathogens such as Clostridium, Bacteroides, Porphyromonas, Prevotella, Histophilus, Shigella and Helcococcus (Jeon and Galvao, 2018; Basbas et al., 2023; Becker et al., 2023). Reports on the detection of bovine uterine pathogens using mPCR are megre. Dong-bo et al. (2011) developed an mPCR for simultaneous detection of S. aureus, E. coli and B. cereus in bovine endometritis, reporting 23.95% triple infections and 17.15% co-infections with S. aureus and E. coli, which is lower than the E. coli detection rate in our study. Similarly, Dung et al. (2024) observed 100% sensitivity for Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and E. coli and 63.6% for Staphylococcus aureus, with specificity ranging from 87.5% to 97.6%. Multiplex PCR, employing multiple primer sets in a single reaction, enables rapid and simultaneous detection of multiple pathogens from a single sample (Zhao et al., 2019; Aziz et al., 2021; Kumar et al., 2021).

Fig 6: mPCR amplification of fimH, C. tertium 16S rRNA and H. somni 16S rRNA gene.



Fig 7: mPCR amplification of fimH, C. tertium 16S rRNA and H. somni 16S rRNA gene.



Table 4: Prevalence, sensitivity, specificity and accuracy of mPCR.

CONCLUSION

In conclusion, the overall prevalence of uterine infection in cattle was recorded 10.45 % whereas E. coli was the most prevalent uterine pathogen in Vindhyan region. Further, a multiplex PCR was developed for simultaneous detection of E. coli, C. tertium and H. somni. in cattle. This method will be helpful to diagnose mixed infection of uterine pathogens in cattle. Further refinement in the methodology may be undertaken to improve the assay. To the best of our knowledge, this is the first report describing the simultaneous detection of E. coli, C. tertium and H. somni from mixed uterine infections. 

ACKNOWLEDGEMENT

The present study was supported by “Faculty Incentive Grant” of Institute of Eminence (IoE), Banaras Hindu University. Authors are highly thankful to Faculty of Veterinary and Animal Sciences and Banaras Hindu University for providing the necessary fund and facilities for undertaking this project.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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    Full Research Article

    Prevalence of Uterine Infections and Development of a Multiplex PCR Assay for their Detection in Cattle

    P
    Priyanka Subhashrao Narwade1
    P
    Priya Ranjan Kumar2,*
    P
    Pesingi Pavan Kumar3
    B
    B. Balamurugan1
    D
    Dayanidhi Jena2
    S
    Sanjay Kumar Ravi1
    R
    Rabindra Mohan Mishra4
    M
    Mayukh Ghosh5
    A
    Anshuman Kumar6
    1Department of Veterinary Gynaecology and Obstetrics, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
    2Unit of Teaching Veterinary Clinical Complex, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
    3Department of Veterinary Public Health and Epidemiology, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
    4Krishi Vigyan Kendra, Chatra-825 401, Jharkhand India.
    5Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.
    6Department of Animal Genetics and Breeding, Faculty of Veterinary and Animal Sciences, Rajiv Gandhi South Campus-Banaras Hindu University, Mirzapur-231 307, Uttar Pradesh, India.

    ABSTRACT

    Background: Uterine infections are a leading cause of infertility and economic loss in dairy cattle, often resulting from complex polymicrobial infections. Rapid and accurate detection of the causative pathogens is essential for effective management. This study focuses on assessing the prevalence of uterine infections and developing a multiplex PCR assay for simultaneous detection of key bacterial pathogens in cattle.

    Methods: A total of 1,905 cattle from the Vindhyan region were screened for uterine discharge and categorized as clear with tiny flakes, cloudy, mucopurulent, purulent, or haemorrhagic. Sixty uterine samples were aseptically collected for molecular analysis. Conventional PCR was employed to detect Escherichia coli, Fusobacterium necrophorum, Trueperella pyogenes, Clostridium tertium and Histophilus somni. A multiplex PCR (mPCR) assay was subsequently developed for simultaneous detection of E. coli, C. tertium and H. somni and its sensitivity, specificity and accuracy were assessed to evaluate diagnostic performance.

    Result: The overall prevalence of uterine infections was 10.44%, consisting of 60.80% subclinical endometritis, 26.63% clinical endometritis and 12.56% metritis. The prevalence rates of E. coli, C. tertium and H. somni were 85%, 92.9% and 73.33%, respectively. Conventional PCR detected E. coli in 54, F. necrophorum in 01, T. pyogenes in 70, C. tertium in 44 and H. somni in 12 of the 60 uterine samples. Using mPCR, 51 samples were positive for E. coli, 44 for C. tertium and 15 for H. somni. The sensitivity, specificity and accuracy of the mPCR were 100%, 66.6% and 95% for E. coli; 100% each for C. tertium; and 80%, 100% and 95% for H. somni, respectively, indicating its high diagnostic efficiency.

    INTRODUCTION

    Efficient reproductive performance is fundamental to profitable dairy farming, as it directly impacts herd productivity. However, uterine infections remain a major constraint, contributing significantly to infertility, subfertility and economic loss. Uterine infections, broadly categorized as endometritis, metritis and pyometra depending on their severity and clinical presentation (Azawi, 2008), impair both fertility and productivity. Their economic impact is substantial, arising from delayed conception, increased insemination frequency, prolonged calving intervals, reduced milk yield and premature culling (Deka et al., 2021). The losses are especially severe for smallholder farmers where livestock constitute a primary source of income and nutrition.
           
    The prevalence of uterine infections in cattle varies considerably, with studies indicating that nearly half of dairy cows experience some degree of uterine infection during the postpartum period (Comlekcioglu et al., 2024). The incidence is notably higher among high-yielding dairy breeds (Várhidi et al., 2024). Subclinical uterine infections, often overlooked during routine herd management, pose a major challenge as they silently impair fertility. Prevalence data, therefore, are critical for designing effective reproductive management programs. Uterine infections in cattle are typically caused by a variety of bacterial pathogens, including Escherichia coli (E. coli),Trueperella pyogenes (T. pyogenes), Fusobacterium necrophorum (F. necrophorum), Prevotella melaninogenica (P. melaninogenica) Staphylococcus aureus (S. aureus), Streptococcus uberis (S. uberis), Bacteroides spp. and Proteus spp. usually as mixed infections (Drillich and Wagener, 2018; Jeon et al., 2018). Clostridium tertium (C. tertium), Histophilus somni (H. somni) and Shigella spp. were less frequently associated with bovine uterine infections in earlier literature, however emerging as potential pathogens of uterine infections. The complex and polymicrobial etiology of uterine infections poses significant challenges for their diagnosis and management. Traditional diagnostic methods, such as clinical examination, vaginal mucus scoring, uterine cytology and bacteriological culture, have long been used in veterinary practice. However, these methods are often constrained by limited sensitivity, delayed results and the inability to clearly distinguish pathogenic organisms from commensals (Barlund et al., 2008).
           
    Recent advances in molecular diagnostics have transformed the understanding of bovine uterine infections. Techniques such as PCR, real-time PCR (RT-PCR), multiplex PCR (mPCR) and sequencing-based methods offer enhanced sensitivity, specificity and rapid detection compared to traditional approaches (Aghamiri et al., 2014; Kumar Chetan et al., 2021; Selim et al., 2024; Tahir et al., 2025). These molecular approaches allow for accurate identification of pathogens, detection of mixed infections and characterization of virulence-associated genes, supporting precise diagnosis. Furthermore, PCR-based assays allow direct detection of bacterial DNA from uterine samples, reduces dependence on viable cultures and facilitates large-scale epidemiological surveillance. These advantages highlight the growing importance of molecular diagnostics in the context of bovine uterine infections. Therefore, the present study was designed to investigate the prevalence of uterine infections in cattle in Vindhyan region of Uttar Pradesh, India and to develop and evaluate the utility of molecular diagnostic approaches in their detection.

    MATERIALS AND METHODS

    Study area and sample collection
     
    A total of 1,905 cattle were screened across the Vindhyan region of Uttar Pradesh, of which, 199 were diagnosed with uterine infections. Among the infected cattle, 60 uterine samples were collected aseptically from Mirzapur, Bhadohi, Varanasi and Sonbhadra districts of Vindhyan region. The uterine fluid was immediately transferred into sterile collection vials. The collected samples were initially preserved at 4°C in 20% glycerol stock to maintain microbial viability and subsequently stored at -20°C until further processing.
     
    Categorization of samples and case definition
     
    The collected samples were categorized based on physical characteristics of the uterine contents, which included clear with tiny flakes, cloudy, mucopurulent, purulent and bloody discharges. Cows exhibiting clear with tiny flakes were categorized as subclinical endometritis. Animals showing cloudy or mucopurulent discharge were categorized as clinical endometritis whereas, cows with purulent or bloody discharge were categorized as metritis.
     
    DNA extraction
     
    Genomic DNA from the sample collected was extracted using a GSure® Bacterial DNA Isolation Kit (GCC Biotech, India) as per the manufacturer’s protocol.
     
    Primers
     
    The nucleotide sequence of E. coli fimH gene, F. necrophorum IlkA gene, T. pyogenes plo gene, C. tertium 16S rRNA gene were retrieved from the NCBI GenBank database and the specific pairs of oligonucleotide primers were designed using primer blast of NCBI. The primers for H. somni 16S rRNA gene was taken from published literature. The designed and published primers were subsequently synthesized commercially at Barcode Biosciences Pvt. Ltd. (Bengaluru, India). The details are presented in Table 1.

    Table 1: Primers pairs designed to amplify target genes.



    Reaction conditions of PCR
     
    PCR conditions were optimized for specific and efficient amplification. Each reaction was performed in a 25 µL volume, containing 12.5 µL of 2X PCR master mix, 1 µL each of forward and reverse primers, 3 µL of diluted DNA template and 7.5 µL of nuclease-free water. The amplification protocol included an initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation, primer annealing and extension. Denaturation was optimized at 95°C for 1 minutes. Annealing temperatures were optimized separately for each primer pair: 65°C for 1 min for the fimH gene, 52°C for 45 s for the IlkA gene, 57°C for 30s for plo gene, 55°C for 30 s for the C. tertium 16S rRNA gene and H. somni 16S rRNA gene. Extension was performed at 72°C for 1 min in each cycle, followed by a final extension at 72°C for 10 min. PCR products (10 µL) were resolved on a 1% agarose gel, stained with ethidium bromide and visualized using a gel documentation system.
     
    Sequencing of amplicon
     
    Representative products were purified and subjected to Sanger sequencing. Chromatograms were analyzed using Chromas (version 2.6.6) software and sequences were compared with GenBank entries using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
     
    Reaction conditions of multiplex PCR
     
    Three bacteria namely E. coli, C. tertium and H. somni were selected for mPCR and reaction conditions were standardized. Each mPCR reaction was carried out in a 25 µL volume, containing 12.5 µL of 2X PCR master mix, 6 µL of primers (1 µL each of forward and reverse primers for each gene), 3 µL of diluted DNA template and 3.5 µL of nuclease-free water. The amplification protocol consisted of an initial denaturation at 95°C for 10 min, followed by denaturation 95°C for 1 min, annealing at 59°C for 1 min and extension at 72°C for 1 min, repeated for 30 cycles in a thermocycler. The final extension was carried out at 72°C for 10 min to ensure complete synthesis of amplicons. Each mPCR product (10 µL) was subjected to electrophoresis on a 1% agarose gel, stained with ethidium bromide and visualized using a gel documentation system.
     
    Ethical approval
     
    All procedures for sample collection and laboratory analysis were performed in accordance with the institutional guidelines for animal experimentation. Ethical clearance was obtained from the Institutional Animal Ethics Committee (IAEC), Faculty of Veterinary and Animal Sciences, Banaras Hindu University, under approval number IAEC/BHU/FVAS/2024/173.

    RESULTS AND DISCUSSION

    Prevalence of uterine infection
     
    Out of a total of 1,905 cattle screened across the Vindhyan region of Uttar Pradesh, 199 animals were diagnosed with uterine infections. The diagnosis was based on a combination of reproductive history, clinical signs and the physical characteristics of the uterine discharge. The overall prevalence of uterine infection was found to be 10.44%. The overall and district-wise prevalence of subclinical endometritis, clinical endometritis and metritis is summarized in Table 2, while the prevalence of E. coli, C. tertium and H. somni in the region is presented in Table 4. These findings indicate a considerable burden of uterine infections among cattle in the region, emphasizing the need for improved reproductive health monitoring and timely diagnosis and therapeutic interventions. Reports show substantial variability in the prevalence of uterine infections in cattle across studies and regions, with some studies reporting rates as high as 50% (Sheldon et al., 2008; Comlekcioglu et al., 2024; Mekibib et al., 2024). The prevalence of metritis has been reported to range from 7.2% to 40% (Várhidi et al., 2024; Nguyen et al., 2025), while clinical endometritis ranges from 5% to 35.5% and subclinical endometritis from 11% to 72% (Galvao, 2012; Aghamiri et al., 2014; Sarkar et al., 2016; Kumar Chetan et al., 2021). The prevalence of E. coli in bovine uterine infections has been reported to range from 33.4% to 72.18% (Bicalho et al., 2010; Dutta et al., 2019; Shafique et al., 2021), whereas literature on the prevalence of C. tertium and H. somni is megre. Variations in reported prevalence rates likely reflect differences in diagnostic methods, criteria for classifying uterine infections, postpartum stages, as well as factors such as parity and herd management practices. (Lewis 1997; Aghamiri et al., 2014). Uterine infections are frequently associated with predisposing reproductive disorders such as dystocia, uterine prolapse, abortion and retained fetal membranes (Bell and Roberts, 2007; Singhal et al., 2011; Honparkhe et al., 2025), which compromise uterine defense mechanisms and facilitate bacterial colonization of the endometrium. These conditions collectively contribute to impaired uterine health and subsequent fertility problems in affected animals.

    Table 2: Prevalence of uterine infection across the vindhyan region.


     
    Development of multiplex PCR assay
     
    In order to develop mPCR assay, conventional PCR was first performed to identify the E. coli, C. tertium, F. necrophorum, T. pyogenes and H. somni as a gold standard test. In the present study E. coli (Fig 1; Table 3) emerged as the predominant uterine pathogen, followed by C. tertium (Fig 4; Table 3) across all categories of uterine discharge. In addition, F. necrophorum (Fig 2; Table 3), T. pyogenes (Fig 3; Table 3) and H. somni (Fig 5; Table 3) were also detected, highlighting the complex microbial interactions contributing to uterine pathology. All the amplicon was confirmed by Sanger sequencing except Plo gene of T. pyogenes which may be due to non-specific amplification. E. coli is often regarded as the primary etiological agent of the uterine infection (Sheldon et al., 2002; Williams et al., 2005; Mekibib et al., 2024), gaining access to the uterine lumen during or shortly after parturition. Subsequently, T. pyogenes and F. necrophorum may establish synergistic infections (Sheldon et al., 2008; Haimerl et al., 2018). The identification of C. tertium and H. somni in the current study is noteworthy, as these organisms are less frequently associated with bovine uterine infections in earlier literature (Olson, 1984; Jeon et al., 2015; Saad et al., 2022; Molín et al., 2024) suggesting potential regional variation in pathogen ecology or identification techniques. The observed variation in bacteriological findings across studies may also be attributed to differences in sampling stage, diagnostic criteria, geographical conditions, animal breed and management practices. Collectively, these findings indicate that while E. coli continue to play a dominant role in uterine infections, emerging bacteria such as C. tertium and H. somni may be gaining epidemiological significance in specific herds or regions, warranting further investigation using molecular and metagenomic approaches.

    Table 3: Findings of uterine pathogens using PCR across the vindhyan region.



    Fig 1: PCR amplification of E. coli (fimH) gene at 604 bp.



    Fig 2: PCR amplification of F. necrophorum (IlkA) gene at 406 bp.



    Fig 3: PCR amplification of T. pyogenes (plo) gene at 1010 bp.



    Fig 4: PCR amplification of C. tertium 16S rRNA gene at 988 bp.



    Fig 5: PCR amplification of H. somni 16S rRNA gene at 313 bp.


           
    Based on PCR and Sanger sequencing result, a mPCR assay was successfully developed for the simultaneous detection of E. coli, C. tertium and H. somni (Fig 6 and 7; Table 4). The developed mPCR accurately identified all three pathogens in 90% (54/60) of samples, while 10% (6/60) were correctly identified for two of the three target pathogens. The assay showed high sensitivity and accuracy, with moderate specificity for E. coli, whereas C. tertium and H. somni exhibited high sensitivity, specificity and accuracy (Table 4). Traditionally, T. pyogenes, E. coli, F. necrophorum and P. melaninogenica are considered the major uterine pathogens (Sheldon and Dobson, 2004; Azawi, 2008); however, recent studies have highlighted emerging pathogens such as Clostridium, Bacteroides, Porphyromonas, Prevotella, Histophilus, Shigella and Helcococcus (Jeon and Galvao, 2018; Basbas et al., 2023; Becker et al., 2023). Reports on the detection of bovine uterine pathogens using mPCR are megre. Dong-bo et al. (2011) developed an mPCR for simultaneous detection of S. aureus, E. coli and B. cereus in bovine endometritis, reporting 23.95% triple infections and 17.15% co-infections with S. aureus and E. coli, which is lower than the E. coli detection rate in our study. Similarly, Dung et al. (2024) observed 100% sensitivity for Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and E. coli and 63.6% for Staphylococcus aureus, with specificity ranging from 87.5% to 97.6%. Multiplex PCR, employing multiple primer sets in a single reaction, enables rapid and simultaneous detection of multiple pathogens from a single sample (Zhao et al., 2019; Aziz et al., 2021; Kumar et al., 2021).

    Fig 6: mPCR amplification of fimH, C. tertium 16S rRNA and H. somni 16S rRNA gene.



    Fig 7: mPCR amplification of fimH, C. tertium 16S rRNA and H. somni 16S rRNA gene.



    Table 4: Prevalence, sensitivity, specificity and accuracy of mPCR.

    CONCLUSION

    In conclusion, the overall prevalence of uterine infection in cattle was recorded 10.45 % whereas E. coli was the most prevalent uterine pathogen in Vindhyan region. Further, a multiplex PCR was developed for simultaneous detection of E. coli, C. tertium and H. somni. in cattle. This method will be helpful to diagnose mixed infection of uterine pathogens in cattle. Further refinement in the methodology may be undertaken to improve the assay. To the best of our knowledge, this is the first report describing the simultaneous detection of E. coli, C. tertium and H. somni from mixed uterine infections. 

    ACKNOWLEDGEMENT

    The present study was supported by “Faculty Incentive Grant” of Institute of Eminence (IoE), Banaras Hindu University. Authors are highly thankful to Faculty of Veterinary and Animal Sciences and Banaras Hindu University for providing the necessary fund and facilities for undertaking this project.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article.

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