Paramphistomosis is an important trematode infection of ruminants caused by members of the family Paramphistomatidae and is widely distributed in warm and tropical regions. The parasite has a complex life cycle involving freshwater snails as intermediate hosts, while metacercariae represent the infective stage for ruminants. Following ingestion, the juvenile flukes undergo a migratory phase through the gastrointestinal tract before reaching their predilection sites in the rumen and reticulum. Although adult flukes in the rumen are often associated with limited clinical manifestations, heavy infections, particularly during the intestinal migratory stage, can cause substantial tissue damage and may lead to hypoproteinemia, anemia and, in severe cases, mortality (Gelberg, 2017). Nevertheless, increasing evidence indicates that adult rumen flukes may also induce local pathological changes at their attachment sites, including alterations of ruminal papillae, epithelial damage and inflammatory responses (Fuertes et al., 2015; Rizwan et al., 2022). More recently, significant reductions in epithelial thickness and ruminal papillary dimensions have also been demonstrated in paramphistome infected ruminants (Rizwan et al., 2025). The rumen plays a central role in ruminant digestion, serving as a major site of microbial fermentation and nutrient absorption. Volatile fatty acids generated by the ruminal microbiota represent an important source of metabolic energy and are absorbed predominantly across the ruminal epithelium (Xiao et al., 2016; Rizwan et al., 2022). The stratified squamous epithelium, characterized by numerous ruminal papillae, is highly specialized for absorption and functions as an important barrier between the ruminal environment and the underlying tissues. Maintenance of its structural and functional integrity is therefore essential for normal ruminal physiology and efficient nutrient utilization (Kahl et al., 2021). Any persistent insult to the ruminal mucosa may compromise epithelial function and consequently affect local tissue homeostasis. Despite recent histopathological investigations of paramphistome infected ruminal tissues (Rizwan et al., 2022; Rizwan et al., 2025), the biochemical and metabolic consequences of these tissue alterations remain poorly characterized. One potential mechanism underlying tissue injury during parasitic infection is oxidative stress, which arises when the generation of reactive oxygen species (ROS) and other reactive molecules exceeds the capacity of the antioxidant defense system to neutralize them. Under physiological conditions, pro-oxidant and antioxidant processes are maintained in a dynamic equilibrium; disruption of this balance can result in oxidative damage to cellular lipids, proteins and nucleic acids (Georgieva et al., 2006). Reactive oxygen species also constitute an important component of the host innate immune response. During infection, activated immune cells generate ROS through the oxidative burst to contribute to the elimination of invading pathogens. However, excessive or sustained ROS production may overwhelm endogenous antioxidant defenses and consequently cause collateral damage to host tissues (Sies, 1997).
       
Trematode infections have been associated with alterations in host redox homeostasis and antioxidant defenses. Tissue invasion, attachment, feeding activity and the associated inflammatory response may promote the production of reactive oxygen and nitrogen species, thereby increasing oxidative stress and lipid peroxidation while reducing antioxidant capacity. Reduced glutathione (GSH), an important intracellular antioxidant, may be consumed during the neutralization of excessive reactive species, further contributing to redox imbalance and tissue injury (Von Bülow et al., 2024). However, although previous studies have characterized the histopathological effects of adult paramphistomes on the ruminal mucosa, little is known about whether these lesions are accompanied by local oxidative imbalance and alterations in major tissue biochemical components. In particular, the combined relationship among ruminal histopathology, oxidative stress and carbohydrate and protein alterations during natural adult Paramphistomum spp. infection remains poorly understood. Therefore, the present study aimed to address this gap by integrating histopathological examination, histochemical assessment of carbohydrates and proteins and oxidative stress biomarkers in the rumen of cattle naturally infected with adult Paramphistomum spp. Malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) were evaluated together with tissue carbohydrate and protein distribution and compared with non-infected ruminal tissues. This integrated approach provides a more comprehensive assessment of the local structural, biochemical and oxidative alterations associated with adult Paramphistomum spp. infection.

Examination procedures and rumen sample collection
 
Rumen and reticulum samples were collected from slaughtered cattle at the slaughterhouse in Jazan City, Saudi Arabia. The study was conducted during 2026 and the laboratory work was carried out at the Parasitology Laboratory, Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia. Following slaughter and complete exsanguination, the rumen and reticulum were removed and examined post-mortem for the presence of adult rumen flukes (Paramphistomum spp.). The rumen was opened longitudinally and the ruminal mucosa was carefully examined for attached flukes, particularly within the dorsal and ventral sacs and areas showing gross pathological changes. Cattle in which adult rumen flukes were detected were considered infected (Fig 1). For histopathological examination, approximately 1 cm² tissue specimens were collected from the ruminal mucosa at sites of parasite attachment and/or visible gross lesions, immediately fixed in 10% neutral buffered formalin and subsequently processed using routine histological procedures (Zitnan et al., 2008). For oxidative stress assessment, separate rumen tissue specimens were collected from infected areas immediately after confirmation of infection. The specimens were placed in sterile, labeled tubes, kept on ice during transportation to the laboratory and stored at -80°C until biochemical analysis. The preserved tissue samples were subsequently used for determination of glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) levels (Miranda et al., 2026).
 
Histopathological and histochemical studies
 
Rumen tissue samples were promptly collected from infected and non-infected cattle and immediately fixed in 10% neutral buffered formalin. For histopathological and histochemical examinations, rumen tissue samples from 20 cattle were used, comprising 10 naturally infected and 10 non-infected cattle. As samples were obtained post-mortem from routinely slaughtered cattle, detailed data on age, health status and feeding conditions were unavailable and infection was classified based on the presence of adult Paramphistomum spp. without quantifying parasite burden. Following fixation, the tissue specimens were washed in running tap water, routinely processed by dehydration through graded concentrations of ethanol, cleared in xylene and embedded in paraffin wax. Serial paraffin sections of 5 μm thickness were obtained using a rotary microtome and mounted on glass slides. For histopathological examination, tissue sections were deparaffinized in xylene, rehydrated through descending concentrations of ethanol and stained with conventional hematoxylin and eosin (H and E) according to Fischer et al., (2008). The stained sections were examined microscopically for histopathological alterations associated with adult Paramphistomum spp. infection.
       
For histochemical assessment, additional 5-μm-thick sections were prepared from the same paraffin-embedded rumen tissues. Carbohydrate-containing components were demonstrated using periodic acid-schiff (PAS) staining according to Hotchkiss (1948), whereas total protein distribution was demonstrated using the mercury bromophenol blue method described by Mazia et al., (1953). All stained sections were examined using a light microscope (Olympus CX41, Olympus Corporation, Tokyo, Japan) and representative fields were photographed using an Olympus digital camera attached to the microscope. Histochemical reactions were evaluated qualitatively based on the intensity and distribution of staining within the different structural components of the rumen tissue and staining intensity was categorized according to the degree of color development observed microscopically.
 
Oxidative status in the rumen of cattle infected with adult Paramphistomum spp.
 
Rumen tissue samples were collected from cattle infected with adult Paramphistomum spp. and processed for the assessment of oxidative stress biomarkers. Oxidative stress analysis was performed on rumen tissues from the same 20 cattle (10 infected and 10 non-infected) used for the histopathological and histochemical examinations. Briefly, representative pieces of rumen tissue were accurately weighed and homogenized at a ratio of 1:10 (w/v) in ice-cold phosphate-buffered saline (PBS; pH 7.4) using a homogenizer under chilled conditions. The tissue homogenates were centrifuged at 5,000 × g for 15 min at 4°C. The resulting supernatants were carefully collected and stored at -80°C until biochemical analysis. The concentrations of reduced glutathione (GSH), nitric oxide (NO) and malondialdehyde (MDA) were determined using commercially available diagnostic kits (BioDiagnostic Co., Giza, Egypt), according to the manufacturers’ instructions. GSH levels were determined according to the method described by Moron et al., (1979), whereas NO levels were estimated following the method of Montogomery and Dymock (1961). Lipid peroxidation was assessed by measuring MDA concentrations using the thiobarbituric acid reactive substances (TBARS) method described by Ohkawa et al., (1979). The oxidative status of the rumen tissue was evaluated based on the levels of MDA as an indicator of lipid peroxidation and oxidative damage, together with GSH as a major endogenous antioxidant and NO as a marker associated with nitrosative/oxidative stress.
 
Statistical analysis
 
Statistical significance was assessed using an independent-samples t-test to compare oxidative stress biomarkers between infected and non-infected groups. Data were expressed as mean±standard deviation (SD). Cohen’s d was calculated to estimate the effect size. All p-values were two-tailed and P≤0.05 was considered statistically significant. Statistical analyses were performed using SPSS.

Histopathological examination revealed distinct alterations in the rumen of cattle infected with adult Paramphistomum spp. compared with non-infected animals. The non-infected rumen exhibited preserved tissue architecture, with prominent and elongated ruminal papillae and an intact stratified squamous epithelial lining. In contrast, infected ruminal tissues showed shortening and blunting of the ruminal papillae, thinning and irregularity of the epithelial lining with focal epithelial erosion and apparent widening of the underlying connective tissue. At higher magnifications (×10 and ×20), inflammatory cell infiltration was clearly observed within the connective tissue beneath the affected epithelium. Collectively, these histopathological findings demonstrate mucosal injury and an associated local inflammatory response in the rumen of cattle infected with adult Paramphistomum spp (Fig 2). These findings are consistent with previous reports of structural alterations in the ruminal mucosa associated with paramphistome infection. Rizwan et al., (2022) observed reduced epithelial thickness and papillary dimensions, epithelial disruption and compromised cell junctions in Paramphistomum infected ruminants. Similarly, Fuertes et al., (2015) described marked alterations of ruminal papillae and epithelial lesions in cattle naturally infected with adult Calicophoron daubneyi. The inflammatory cell infiltration observed in the present study also agrees with that reported during experimental Paramphistomum cervi infection (Singh et al., 1984) and at sites of adult fluke attachment (Fuertes et al., 2015). Such epithelial and inflammatory changes may result from persistent parasite attachment and mechanical irritation, potentially impairing the protective and absorptive functions of the ruminal mucosa. Overall, these observations support the pathological significance of adult Paramphistomum spp. infection in the rumen.


       
Histochemical examination revealed marked alterations in both carbohydrate and protein contents in the ruminal tissues of cattle infected with adult Paramphistomum spp. Periodic acid-Schiff staining showed a strong purple reaction in non-infected rumen sections, indicating abundant carbohydrate-containing components, whereas infected tissues exhibited markedly weaker staining, indicating reduced carbohydrate content. Similarly, mercuric bromophenol blue staining revealed a relatively strong blue reaction in non-infected ruminal tissues, while infected sections showed weaker and more diffuse staining, consistent with reduced protein content. Collectively, these histochemical findings demonstrate a concurrent reduction in carbohydrate- and protein-associated staining in Paramphistomum spp. infected ruminal tissues (Fig 3 and 4). The concurrent reduction in carbohydrate- and protein-associated staining observed in Paramphistomum spp. infected ruminal tissues indicates substantial alterations in the nutrient composition and metabolism of the affected tissue. This finding is consistent with Sahai et al., (1985), who reported reductions in polysaccharides, glycogen and protein in the intestinal tissues of goats experimentally infected with P. cervi, suggesting that paramphistome infection can alter host-tissue nutrient reserves.




       
The reduction in carbohydrates may be particularly related to their utilization as energy substrates, as glucose and glycogen play important roles in the metabolism of rumen amphistomes (Bahadur and Gupta, 1984). Recent molecular studies have further demonstrated active glycolytic and glycogen metabolic pathways in the rumen fluke Calicophoron daubneyi (Huson et al., 2021), supporting the high carbohydrate requirements of these parasites. In contrast, the reduction in tissue protein may reflect a combination of parasite nutrient acquisition and local tissue alterations associated with parasite attachment and feeding activity. Recent genomic and molecular evidence from C. daubneyi has identified specialized mechanisms related to feeding, nutrient acquisition and host-parasite interactions (Huson et al., 2021; Clancy et al., 2025). Similar reductions in host carbohydrate and/or protein reserves have also been described in other parasitic infections, including Fasciola hepatica, schistosomes and Eimeria stiedae (Lloyd and Barrett, 1983; El-Sheikh and Nagi, 1991; Mares et al., 2026). Taken together, the simultaneous reduction in carbohydrate and protein contents observed in the present study may reflect the combined effects of parasite nutrient utilization, altered host-tissue metabolism and local pathological damage associated with Paramphistomum spp. infection.
       
The oxidative stress status of ruminal tissues was assessed by measuring nitric oxide (NO), glutathione (GSH) and malondialdehyde (MDA) Table 1. As shown in Fig 5, NO concentration was significantly higher in infected tissues (18.55±5.47 µmol/g tissue) than in non-infected tissues (8.60±5.15 µmol/g tissue) (t = 2.96, P = 0.018; Cohen’s d = 1.87).



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The increased NO level may reflect enhanced inflammatory and nitrosative responses associated with parasitic infection, as inflammatory activation can stimulate NO production in infected tissues (Bogdan, 2001; Wink et al., 2011).
       
In contrast, GSH concentration was significantly lower in infected tissues (6.90±3.45 mg/g tissue) compared with non-infected tissues (15.85±7.53 mg/g tissue) (t = -2.41, P = 0.042; Cohen’s d = -1.53) (Fig 6). As an important intracellular antioxidant, GSH contributes to maintaining cellular redox balance; therefore, its depletion may indicate increased utilization of antioxidant defenses under oxidative challenge (Forman et al., 2009). Similar alterations in antioxidant status have been reported in sheep naturally infected with the trematode Fasciola hepatica (Saleh, 2008).


       
MDA concentration was significantly increased in infected tissues (22.79±5.76 nmol/g tissue) compared with non-infected tissues (12.46±1.55 nmol/g tissue) (t = 3.87, P = 0.005; Cohen’s d = 2.45) (Fig 7). This difference remained significant in the Welch’s t-test (P = 0.014). Since MDA is a marker of lipid peroxidation, its elevation suggests increased oxidative damage to cellular membranes (Ayala et al., 2014). Similar increases in MDA, accompanied by alterations in antioxidant defenses, have been reported in animals infected with F. hepatica (Saleh, 2008; Nasreldin and Zaki, 2020). Similarly, alterations in oxidative stress biomarkers, including GSH, MDA and NO, have been reported during Eimeria stiedae infection, further supporting the association between parasitic infection and oxidative imbalance (Mares et al., 2024).


       
Taken together, the increased NO and MDA levels and reduced GSH concentration indicate a significant disturbance in the redox balance of Paramphistomum infected ruminal tissue. This oxidative/nitrosative imbalance may be associated with local inflammation and tissue damage caused by parasite attachment and infection. Histopathological studies of paramphistome infected rumen have reported epithelial degeneration and reduced epithelial thickness, supporting the occurrence of structural alterations in infected ruminal tissue (Rizwan et al., 2025). Therefore, the biochemical changes observed in the present study may contribute to, or occur concurrently with, the tissue and histochemical alterations associated with Paramphistomum infection.
       
By integrating histopathological, histochemical and oxidative stress assessments, the present study provides complementary evidence of the local structural and metabolic alterations associated with adult Paramphistomum spp. infection. However, the use of naturally infected slaughterhouse animals limited control over age, health status and feeding conditions, while the absence of parasite burden quantification prevented assessment of the relationship between infection intensity and tissue alterations. Moreover, the qualitative histochemical assessment limits conclusions regarding the magnitude of carbohydrate and protein changes. Further studies incorporating quantified parasite burdens and quantitative histochemical approaches are needed to clarify these relationships and their implications for ruminal function.

The present study demonstrated that Paramphistomum infection is associated with significant histochemical and oxidative alterations in ruminal tissue. Infected tissues showed reduced carbohydrate and protein contents, together with increased NO and MDA levels and decreased GSH concentration. These findings indicate disturbed tissue metabolism and enhanced oxidative/nitrosative stress associated with parasitic infection. Overall, Paramphistomum infection may compromise the metabolic and structural integrity of the ruminal tissue through altered nutrient utilization and oxidative damage.

This work was funded by Ongoing Research Funding Program, (ORF-2026-2200), King Saud University, Riyadh, Saudi Arabia.

The authors declare that they have no conflicts of interest related of this study.