Research on acute phase proteins and inflammatory cytokines biomarkers in dromedary camels (camelus dromedarius) with clinical endometritis

doi:10.21203/rs.3.rs-1495030/v1

Download PDF

Research Article

Research on acute phase proteins and inflammatory cytokines biomarkers in dromedary camels (camelus dromedarius) with clinical endometritis

https://doi.org/10.21203/rs.3.rs-1495030/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Uterine diseases are prevalent in she-camel and lead to economic losses because of decreased fertility. The aim of this study is to look into the expression pattern of acute-phase proteins (APPs) and inflammatory cytokines in dromedary camels with clinical endometritis (CE) to highlight their role in immune-pathogenesis of the disease. Moreover, to identify the use of these parameters as a complementary tool for CE screening as well as investigate the efficacy of ceftiofur antibiotic. APPs and inflammatory cytokines were estimated in camels with CE. Values of APPs (Hp, SAA, and Fg), pro-inflammatory (IFN-γ, TNF-α, IL-1α, IL-1β, and IL-6) and ant-inflammatory cytokines (IL-10) were higher in camels with CE than that of healthy control (P<0.05). The highest correlation was observed between HP and IFN-γ (r = 0.73), and IL-1β and IL-6 (r = 0.73), while the lower correlation was observed between Fg and IFN-γ (r = 0.25). Corynebacterium pyogenes and Arcanobacterium pyogenes were the most common pathogens involved in the etiology of CE. All investigated biomarkers demonstrated a high degree of recognition between CE camel and healthy controls (AUC was > 0.90). A higher proportion of camels with CE that were treated with ceftiofur (90%, P<0.0001) scored clinical cure after the first dose, while 10% required a second dose. In conclusion, CE causes increased APPs and inflammatory cytokines biomarkers indicating a significant acute phase response in diseased camels with CE. These changes in biomarkers could be beneficial for understanding immune-pathogenesis of CE in dromedary camels, clinical practice, and basic clinical research.

Haptoglobin

Serum amyloid A

inflammatory cytokines

Camel

Endometritis

Several infectious diseases and pathological lesions in the female genital tract result in an infertility that is one of the major causes of economic losses in many mammalian species, including dromedary camels. The popularity of pathological lesions among reproductive organs had differences, where the highest proportion was observed in the uterus (Belina et al. 2021). Endometritis is one of the most common lesions, causing decreased fertility and high economic losses (Wernerry and Kumar 1994). The detection of uterine infection is pivotal in controlling the dissemination of infection to other animals through sexual transmission. It is well known that the infection induces a series of inflammatory responses that play key roles in the development and persistence of many pathological states.

The acute-phase response is defined as a complicated process that enclose a broad range of pathophysiological reactions such as muscle protein depletion, changes in hormones, alterations in plasma proteins concentrations, mainly synthesized in the liver, which are called acute-phase proteins (APPs) (Gabay and Kushner 1999). APPs such as serum amyloid A (SAA), haptoglobin (Hp), and fibrinogen (Fg) are important for catching of microorganism and their products, for neutralizing enzymes, and for scavenging reactive oxygen species (ROS), consequently, reduce tissue damage and improve tissue repair (Jain et al. 2011). APPs production is induced by pro-inflammatory cytokines acting as messengers between the local site of infection and the liver cells synthesizing the APPs (Jain et al. 2011).

Pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1α, IL-1β, and IL-6) are produced by immune cells such as helper T cells (Th) and macrophages, and are involved in the immunoprotection as well as in the up-regulation of inflammatory reactions (Zhang and An 2007). The anti-inflammatory cytokines are a series of immunoregulatory molecules that control the proinflammatory cytokine response and act in concert to regulate the body immune response (Opal and DePalo 2000). Among all the anti-inflammatory cytokines, IL-10 is high potent anti-inflammatory cytokine that inhibit the expression of pro-inflammatory cytokines such as TNF-α and IL-6 by activated macrophages (Kevin et al. 2008). The balances of pro- and anti-inflammatory cytokines are essential to inhibit immunopathological disorders.

To be a predictor of disease, a biomarker must be approved, where; the approval criteria incorporate inborn qualities such as specificity, and sensitivity (Dalle-Donne et al. 2006). The foremost instinctive objectives for a biomarker are to analyze symptomatic and presymptomatic infection, and the effectiveness of the perfect biomarker lies in its capacity to supply early signs of infection and/or its development (Andrei et al. 2016).

The inflammatory cytokines and APPs can be used as a diagnostic tool in several bacterial, parasitic and viral diseases (El-Deeb 2013; El-Deeb and Elmoslemany 2016; El-Deeb and Buczinski 2015; El-Deeb et al. 2019; El-Deeb et al. 2020; El-Deeb et al. 2021; Eckersall et al. 2006; Heegaard et al. 2000; Meade et al. 2009). Thus, the aims of the current study are to determine the role of these biomarkers in the pathogenesis of CE, to identify the use of these parameters as a complementary tool for CE screening as well as investigate the efficacy of ceftiofur antibiotic.

Ethical approval

All experimental procedures used in the current study were approved by the guidelines of Animal Ethics Committee at King Faisal University, Saudi Arabia (CVM-2154).

Animals and endometritis diagnosis

A total of 60 she-camels were used in this study. She-camels were clinically investigated in the Veterinary Teaching Hospital (VTH), and Camel Research Center (CRC), King Faisal University, Saudi Arabia. The soundness of animals was evaluated. She-camels without systemic lesions or deformities were classified as suitable for the experiment, and those showing alterations or pathological lesions were excluded. According to clinical examination, and the laboratory analysis, the camels were categorized into two groups. The she-camels of the first group were comprised of healthy individuals (n = 20; control group) however the other group were she-camels with clinical endometritis of different grades (n = 40; Endometritis group). The selection of the control group was based on full clinical examination of she-camels and special clinical examination of the uterus. The healthy she-camels were examined in the VTH and CRC for a routine examination before breeding season. Figure 1 displayed the experimental design of the study. The flowchart showing study design, number of she-camels with and without clinical endometritis.

For each she-camel, the genital tract was examined through rectal palpation and by trans-rectal ultrasound (7.5 MHz transducer, Aloka, Japan) with manual vaginal exploration. The camels were diagnosed with endometritis following criteria described by Sheldon et al. (2006). These criteria include vaginal mucopurulent/purulent discharge, the uterus enlarged and thickened with intrauterine fluid (Catarrhal, mucopurulent, or purulent) accumulation that contains echogenic particles. The camels without endometritis were considered healthy control.

The vaginal discharge was graded using a 0 to 2 scale: 0 = normal uterine discharge; 1, moderate = turbid mucus or turbid mucus with flakes of purulent exudates in the uterine discharge; 2, sever = the uterine discharge is made up of purulent exudates (adapted from Sheldon et al. 2006). Clinical endometritis was defined as any she-camel presenting a score of 1 or 2 (mucopurulent or worse vaginal discharge) at the time of examination.

For bacteriological examination, uterine swabs were collected by passing a sterile double-guarded uterine culture swab (Minitüb, Tiefenbach, Germany) through the cervix, guided by a gloved arm, rotated to the right and left over the uterine wall. Afterward, the swab was retracted into the protecting tube and removed from the animal. Swabs were then broken into sterile 2 mL microfuge tubes containing 1% peptone water (Oxoid, UK).

Biomarkers analysis

Acute-phase proteins (APPs): Test kits (Tridelta Development Ltd., Kildare, Ireland) were used to measure haptoglobin (HP) and serum amyloid A (SAA) in serum samples.

Cytokines (CYTs): In order to estimate CYTs concentrations (TNF-α, IL-1α, IL-1β, IL-6 and IL-10) in serum, commercial ELISAs (MyBioSource, San Diego, USA) were used according to the manufacturer's recommendations. Fibrinogen (Fg) levels were analyzed in plasma samples using the heating precipitation method according to the method described by Thomas (2000).

Bacteriological examination

Each swab was streaked onto MacConkey agar, sheep blood agar, Salmonella-Shigella agar (Oxoid, England), and incubated at 37 ^oC for 48 hours. Bacterial colonies were examined for their morphology, macroscopic characteristics, Gram staining, catalase, and oxidase activities. Colonies were purified on brain heart infusion agar (Oxoid, England) and subjected to biochemical identification by the automated Vitek compact system using GP, GN, and AN identification kits (bioMérieux, France).

Ceftiofur treatment

The previous study in she-camel (Kandeel et al. 2021) reported that one dose of ceftiofur crystalline acid-free form (Ceftiofur-CAF; i/m) at a dose rate of 6.6 mg/kg attained serum level above 0.2 μg/ml for 7 days, and a single dose gives 7 days cure against highly susceptible bacteria. Consequently, treatment was carried out by using a dose 6.6/ mg/kg BW (i/m) of Ceftiofur-CAF 200 mg/ml suspension (Excede, Zoetis Inc, NJ, USA). The response to treatments was assessed on days 7 and 14 d post-therapy administration at the second examination for she-camel with CE. The clinical resolution was defined as the absence of mucopurulent or worse uterine discharge at the second examination i.e. animals with clear, translucent, or no mucus. Clinical cure or failure to cure was determined. The second dose of ceftiofur-CAF after 7 days for those animals did not respond to the first injection.

Statistical analysis

All parameters were expressed as mean ± SD. Comparisons in mean were performed by Kruskal–Wallis ANOVA on Ranks followed by Dunn’s multiple comparisons. The t-test was used to measure the differences between camel with and without endometritis. Statistical analysis was performed using JMP software version 11.0.0 (SAS Institute, Cary, NC, USA). Graphpad Prism v5 software (Graphpad Software, Inc., San Diego, CA) was used to draw the figures. Spearman’s rank correlation test was used to evaluate the correlation between parameters. Each assay’s diagnostic accuracy was evaluated by creating the ROC (receiver operator characteristics) curve and determining the area under the curve (AUC). An AUC of 0.7 to 0.9 was considered moderately accurate, an AUC of >0.9 highly accurate, and an AUC of 1 perfect (Gardner and Greiner 2006). The different means were significant at P<0.05

Expression pattern of acute phase proteins and inflammatory cytokines biomarkers

The values of Hp, SAA, and Fg were higher in camels with endometritis than that of healthy control (P<0.05) (Fig. 2). In addition, the mean values of inflammatory cytokines (IFN-γ, TNF-α, IL-1α, IL-1β, IL-10, and IL-6) were luxuriously higher in the endometritis group (P<0.05) compared with the healthy control (Fig. 3).

Spearman's correlation coefficient matrix in camels with and without endometritis was used to analyze the correlation coefficients (r) between biomarkers (Table 1). The highest correlation was observed between HP and IFN-γ (r = 0.73), and IL-1β and IL-6 (r = 0.73), while the lower correlation was observed between Fg and IFN-γ (r = 0.25).

The biomarkers tested did not distinguish between the bacterial infections, with no differences (P > 0.05) observed between endometritis pathogen-species and changes in biomarker expression (Fig. 4 and 5).

The ROC curves were created, and the AUC was calculated, in order to assess the parameter's accuracy in distinguishing between camels with and without endometritis (Fig. 5 and 6). All pro-and anti-inflammatory cytokines showed high sensitivity and specificity (AUC > 0.90) for CE cases. IL-6 and IFN-γ showed higher sensitivity and specificity compared to the other biomarkers (AUC > 0.96). In addition, HP and SAA had higher sensitivity and specificity (AUC ≥ 0.95) compared with Fg (AUC < 0.90). The optimal cut-off value of IL-6 and IFN-γ when using ROC analysis to distinguish between camels with and without endometritis were 115.80 pg/mL, and 47.30 pg/mL, respectively (Table 2).

Uterine bacterial isolates

The bacteria that colonize the uterus of camels with endometritis are shown in Table 3. The most prominent bacterial isolates from the uterus were made up of 20 % Corynebacterium pyogenes (C. pyogenes) and 12.5% Arcanobacterium pyogenes (A. pyogenes).

Effect of ceftiofur antibiotic

The rates of recovery of clinical signs were high for both initial and second treatments. A higher proportion of she-camel with CE that were treated with ceftiofur (90%, P<0.0001) scored zero (clear mucus; clinical cure) after the first dose, while 10% required a second dose of ceftiofur (Fig. 8).

Cytokines are regulators of the animal's responses to infection, inflammation, and trauma. Some cytokines make the disease worse (pro-inflammatory; IFN-γ, TNF-α, IL-1α, IL-1β, and IL-6), while others reduce the inflammation and enhance healing (anti-inflammatory; IL-10) (Kevin et al. 2008). The present study is considered the first report on the levels of pro-and anti-inflammatory cytokines and APPs in serum of dromedary camels with CE.

IL-6 is a cytokine derived from T cells, produced by lymphocytes, macrophages, and endothelial cells, and inhibits the growth of endometrial cells (Dmowski and Braun 2004). The cytokine TNF-α is produced by activated macrophages, epithelial, glandular epithelial and endothelial cells in the stromal layer of the bovine uterus (Okuda et al. 2010). In this study, serum IL-6 and TNF-α concentrations were higher in CE camels compared to those without CE. Moreover, there was a positive correlation between TNF-α and IL-6 since TNF-α enhances the production of other pro-inflammatory cytokines, such as IL-6, and additional TNF-α (Bhadaniya et al. 2019; Lu et al. 2013). In addition to, TNF-α is involved in the normal physiology of endometrial proliferation and shedding (Lu et al. 2013).

The high concentration of IL-6 in serum of diseased camels may be due to the cell's secretion of this protein in response to the recognition of a pathogen- associated molecular patterns (PAMP) by specific Toll-like receptors (TLR), as described by Sheldon et al. (2009) and Turner et al. (2012). As a result of this reaction, neutrophils and macrophages already present in the uterus become more effective at phagocytosis and enhance the local immune response in infected camel uterus against endometritis pathogens. A study by Singh et al. (2008) found that leucocytes in the uterus and those flowing into it through blood activation or directly stimulated by microorganism PAMPs (mainly phagocytes) produce cytokines, contributing to an increase in their concentration. These responses were evident in our investigated camels with CE by detecting elevated levels of IL-6 and TNF-α.

IL-1β is secreted by mononuclear cells in response to infections (Dinarello 1991). It is known that IL-1β contributes to local effects such as fibrosis, tissue matrix disintegration, and the inflow of inflammatory cells to commence the inflammatory process (Dinarello 2005). The microbial products and endotoxins are strong stimuli for IL-1β (Dinarello 1991), which may explain the increased serum concentrations of IL-1β in investigated camels with CE compared to normal ones.

The IL-1α is basically located in nearly all healthy cell types but is released upon necrotic cell death as a bioactive mediator (Paolo1 and Shayakhmetov 2016). The inflammatory characters of IL-1β are popular knowledge in the biomedical studies, and many are familiar with molecular mechanisms of IL-1β production, however, the role of IL-1α in the pathogenesis of inflammation needs further investigation, because IL-1α is seldom seen in the serum of infected body with inflammatory diseases (Cavalli et al. 2021). In addition to, IL-1α and IL-1β bind the same receptor and induce identical pro-inflammatory effects, however, IL-1α is local, IL-1β is systemic (Rider et al. 2013; Cavalli et al. 2021).

IL-1β, IL-6, and TNF-α are involved in muscle protein degradation and lipolysis. Moreover, the anabolic effect of insulin on skeletal muscle is inhibited by IL-1β (Klasing et al. 1987). Therefore, IL-1β, IL-6, and TNF-α form a network involved in the degradation and lipolysis of muscle proteins and the synthesis APPs in the liver cells (Richards et al. 1991). Inflammatory responses are clearly integrated in this manner as perceived in this study in camels with CE.

By inhibiting gastric motility, gastric emptying, and acid secretion, IL-1β acts centrally to induce anorexia (by acting on neurotransmitters). Additionally, it affects the endocrine system, resulting in altered levels of corticotrophin-releasing factor, cholecystokinin, glucagon, and insulin (Plata-Salaman and Borkoski 1993). During the acute phase of the disease, these central and peripheral actions of leukocytosis can result in a 50% reduction in feed intake. There might be a direct effect of cytokine-mediated neuroendocrine changes on camels with CE and systemic signs.

Researchers have previously reported that IL-1α stimulates endometrial cells to produce IL-6 and IL-8, and the cells' responses are dependent on the concentration of IL-1α (Laura et al., 2015). In addition to inducing inflammatory responses in bovine endometrial cells, IL-1α also stimulates endometrial cells' endocrine function, which results in an increased level of prostaglandin F2α and E2 (Tanikawa et al., 2005; Leung et al. 2001). Herein, a significant elevation in level of IL-1α and IL-1β were seen in camels with CE than normal ones.

IFN-γ has an important role in recognizing and eradication pathogens. It can exhibit its immunomodulatory effects by enhancing antigen processing and presentation, increasing leukocyte trafficking, inducing an anti-microbial functions and affecting cellular proliferation and apoptosis (Kak et al. 2018). Our results showed a significant increase of IFN-γ in CE group than control.

It's extensively appreciated that numerous of the serious complications of infection result from extreme immune activation (Li et al. 1999). Remedial and preventative strategies to enhance immune-mediated clearance of pathogens, and the maximal pathogen control does not accordingly lead to minimal disease and the essential role for immunoregulatory cytokines of the immune response in restricting pathology. IL-10 is an anti-inflammatory cytokine that can act directly on T cells, inhibiting proliferation and production of IFN-γ and TNF-α (Moore et al. 2001). During infection, it inhibits the activity of T helper cells and macrophages, all of which are needed for excellent pathogen clearance but also contribute to tissue damage, hence, IL-10 can both retard pathogen clearance and mitigate immunopathology (Kevin et al. 2008). The control of pro-inflammatory responses to prevent extreme immune activation by microorganisms depends on anti-inflammatory mediators as IL-10 (Couper et al. 2008). In the present study, a significant high concentrations of IL-10 was observed and a positive correlation between IL-10 and pro-inflammatory cytokines concentrations was also detected.

The previous results are consistent with previously reported data by Brodzkia et al. (2015) who reported that the cows with clinical endometritis had higher cytokines levels in uterine washings than those in healthy animals. Moreover, high levels of IL-10 in cows with subclinical endometritis may contribute to the weakening of local resistance mechanisms of the uterus and lead to the persistence of the inflammation. Besides, the inflammatory cytokines were significantly higher in CE of buffaloes as compared to normal uteri (Bhadaniya et al. 2019). Comparable to our findings, Nasreldin et al. (2020) concluded that serum pro-inflammatory cytokines IL-6 and TNF-α are diagnostic markers for CE in ewes and serve as a criterion for different inflammatory complications in ewes classified as having CE.

Pro-inflammatory cytokines are strong stimulators of the generation of APPs, such as SAA, Hp, and Fg (Kim et al. 2014). APPs help in the elimination of infection by modulation of other vulnerable immune proteins or stimulation of phagocytosis. The defensive feature of APPs towards the dangerous outcomes of enzymes fashioned throughout inflammatory reaction, which could result in organ harm, is likewise of big importance (Brodzki et al. 2015b).

In response to uterine infections caused by microbes, APPs are produced in the liver, and their concentration in the serum increases over the first few weeks after birth (Sheldon et al. 2001; Tothova et al. 2008). The synthesis of APPs could occur outside the liver, but their presence in the cells of the uterine endometrium of cows was not demonstrated in vitro (Davies et al. 2008). Nevertheless, there is evidence that uterine endometrium cell production of SAA in cows is possible, based on research conducted by Chapwanya et al. (2013).

We found that HP, SAA, and Fg differ significantly between camels with endometritis and camel free of the disease. SAA, as a protein of the first line of response, is the major APPs that is a rapidly responding, very sensitive marker of inflammation, and its secretion is dependent on TNF-α (Cray et al. 2009; Tothova et al. 2014). While Hp is a minor-to-moderate APPs of the second line of response, whose production is regulated by IL-6, and its increase is slower and characteristic of chronic inflammation (Petersen et al. 2004; Cray et al. 2009; Tothova et al. 2014). Our results appear to confirm these relationships, as the SAA values were significantly higher in the serum of camels with CE compared to healthy controls. Concurrent, both TNF-α and Hp were increased significantly in camels with CE. Such alterations in the APPs concentrations can suggest persistence of chronic inflammation of the uterus. In addition, our results are in accordance with the previous study, which reported increased APPs concentration in the blood serum of cows in response to uterine infection caused by microorganisms (Brodzkia et al. 2015).

Camel's Fg is one of its minor APPs. Coagulation cascades are activated by tissue injury, which involves many different types of substances. In our study of she-camel with CE, Fb synthesis increased during the APR (Davalos and Akassoglou, 2012). Additionally, fibrin also plays a role in inflammation and wound healing, serving as a substrate for fibrin formation (Raynes 1994; Thomas 2000; Murata et al. 2004). As a result, it binds specifically to the CD11/CD18 receptors on the surface of migrated phagocytes, causing increased degranulation, phagocytosis, antibody-dependent cytotoxicity, and delayed apoptosis (Rubel et al. 2001; Murata et al. 2004). Like our results, Hirvonen and Pyorala (1998) and Medcalf (2007) found increased Fg levels under different inflammatory conditions. The presence of high concentrations of APPs in the serum of camels with endometritis is very interesting from a clinical point of view. This protein can be considered a diagnostic indicator of endometritis in camel.

ROC evaluation is a precious approach to assess diagnostic checks and is used to quantify accuracy. ROC analysis was used to evaluate the ability of APPs and cytokines biomarkers to distinguish between camel with CE and healthy controls. The APPs and inflammatory cytokines biomarkers showed a high level of discrimination between CE and healthy control, which is consistent with the guidelines of diagnostic accuracy (Mandrekar 2010). IL-6 and IFN-γ showed higher sensitivity and specificity compared to the other biomarkers (AUC > 0.96). Therefore, they may be considered as an additional tool for diagnosing endometritis in camels.

In the current study, good correlations between APPs and inflammatory cytokines biomarkers were investigated, which resulted in supposing the circumstances that could have happened in the body of a she-camel with CE. Hence, the determination of APPs in she-camels with CE is beneficial for the estimation of damage caused by infection.

The isolated bacteria in camel with endometritis are variable relying on the study. In the current study, the prominent pathogens involved in the etiology of endometritis was C. pyrogens followed by A. pyrogens. These differences in isolated bacteria have been previously suggested to be due to the different geographic locations of the animals included in the studies, the differences in management, the provision of veterinary intervention, the different populations of sampled animals, and the exposure to different antimicrobial drugs (Davis et al. 2013).

Ceftiofur is a β-lactam antibiotic that combines with the bacterial enzyme dd-transpeptidase, blocking the formation of crosslinks between this enzyme and peptidoglycan, which is pivotal for the formation of a rigid cell wall synthesis during bacterial binary fission. Disruptions in cell wall synthesis resulting in cell lysis and the death of the bacteria (Chambers 2007). Ceftiofur has great potency against gram-positive and gram-negative bacteria and is resistant to β-lactamase, therefore blocking the action of these enzymes on the degradation of the β-lactam ring, which deactivates many antibiotics of this class (Hornish and Kotarski 2002). The treatment of CE with ceftiofur antibiotics in she-camel has not been published in recent literature. The current study shows the effectiveness of ceftiofur in eliminating uterine pathogens in she-camel with CE, herein, 90% responded to a single dose of ceftiofur, while 10% from the final administration of a first dose treatment did not ensure resolution and required a second dose. These results are in agreement with Galvao et al. (2009) reported that intrauterine infusion of ceftiofur positively affected uterine health in dairy cows. Additionally, the administration of ceftiofur parenterally has an efficacious therapy for the treatment of acute postpartum metritis in dairy cows (Chenault et al. 2004). Furthermore, because the infection involves deeper layers of the uterus in CE and other genital tissues, systemic therapy would be necessary.

Taking together, the inflammatory cytokines having higher diagnostic importance in uterine inflammation. During endometritis, there is an immune-inflammatory reaction that activates immune cells, producing high amounts of cytokines. With a better understanding of the disease's mechanisms, current treatments can be improved and new remedies can be developed.

In conclusion, the camels with clinical endometritis have remarkable changes in serum APPs, as well as inflammatory cytokines biomarkers indicating a strong acute phase response of CE diseased camels, whereas, we observed higher levels of these biomarkers in camels with CE than healthy controls. Furthermore, there was no significant difference between the levels of examined biomarkers between bacterial isolates infected endometritis camels. These biomarkers may be used to investigate the efficacy of existing medications, understanding the immune-pathogenesis of CE, and to improve new medications in dromedary camels.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author.

Declaration of Interests:

We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported

Funding Statement

This work was supported by [Al Bilad Bank scholarly chair for food security in Saudi Arabia], the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. Chair55]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Andrei S., Matei S., Rugina D., Bogdan L., Ştefănuţ C. Interrelationships between the content of oxidative markers, antioxidative status, and somatic cell count in cow’s milk. Cz. J. Anim. Sci. 2016;61:407–13.
Belina D, Eshetu, A, Alemu S, Shasho B, Mohammed T, Mohammed A, Mummed B, Regassa, D. Reproductive Diseases and Disorders of Female Camels: An Assessment and Pathological and Bacteriological Study in Eastern Ethiopia. Vet. Med. Int. 2021; 13:6641361.
Bhadaniya AR, Prasad MC, Savsani HH, Kalaria VA, Fefar DT, Mathpati BS, Javia BB. Pro-inflammatory cytokine expression studies of subclinical and clinical endometritis in endometrial tissues of buffaloes. Trop Anim Health Prod. 2019; 51(5):1161–66.
Brodzki P, Kostro K, Krakowski L, Marczuk J. Inflammatory cytokine and acute phase protein concentrations in the peripheral blood and uterine washings of cows with subclinical endometritis in the late postpartum period. Vet Res Commun. 2015; 39(2):143–49.
Brodzkia P, Kostrob K, Brodzkic A, Wawrona W, Marczukd J, Kurekd L. Inflammatory cytokines and acute-phase proteins concentrations in the peripheral blood and uterus of cows that developed endometritis during early postpartum. Theriogenology, 2015;84(1):11–18.
Cavalli G, Colafrancesco S, Emmi G, Imazio M, Lopalco G, Maggio MC, Sota J, Dinarello CA. Interleukin 1α: a comprehensive review on the role of IL-1α in the pathogenesis and treatment of autoimmune and inflammatory diseases. Review Autoimmun Rev. 2021;20(3):102763.
Chambers, H.R., Beta-lactam and other cell wall and membrane-active antibiotics. in: Katzung B.G. Basic and Clinical Pharmacology. 10th. McGraw Hill Medical, New York, NY2007: 726–744.
Chapwanya A, Meade KG, Doherty ML, Callanan JJ, O’Farrelly C, 2013: Endometrial epithelial cells are potent producers of tracheal antimicrobial peptide and serum amyloid A3 gene expression in response to E. coli stimulation. Vet Immunol Immunopathol 151, 157–162.
Chenault, J. R., McAllister, J. F., Chester, T., Dame, K. J., Kausche, F. M., & Robb, E. J. (2004). Efficacy of ceftiofur hydrochloride administered parenterally for the treatment of acute postpartum metritis in dairy cows. J. Am. Vet. Med. Assoc. 224, 1634–1639.
Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. J Immunol. 2008;180(9):5771–77.
Cray C, Zaias J, Altman NH. Acute phase response in animals: A review. Comp. Med. 2009;59:517–26.
Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A. Biomarkers of Oxidative Damage in Human Disease. Clinical Chemistry 2006; 52(1):601–23.
Davalos, D. & K. Akassoglou, 2012. Fibrinogen as a key regulator of inflammation in disease. Seminars in Immunopathology, 34, 43–62.
Davies D, Meade KG, Herath S, Eckersall PD, Gonzalez D, White JO, Conlan RS, O’Farrelly C, Sheldon IM, 2008: Toll-like receptor and antimicrobial peptide expression in the bovine endometrium. Reprod Biol Endocrinol 6, e53.
Di Paolo1 NC, Shayakhmetov DM. Interleukin 1α and the inflammatory process Nat Immunol. 2016;17(8):906–13.
Dinarello CA. Blocking IL-1 in systemic inflammation. J Exp Med. 2005;201:1355–59.
Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood. 1991;77(8):1627–52.
Dmowski WP, Braun DP. Immunology of endometriosis. Best Pract Res Clin Obstet Gynaecol. 2004;18:245–63.
Eckersall, P.D.; Young, F.J., Nolan, A.M., Knight, C.H., McComb, C., Waterston, M.M., Hogarth, C.J., Scott, E.M. & Fitzpatrick, J.L. (2006). Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis. Journal of Dairy Science Vol. 89, No. 5, (May 2006), pp. 1488–1501, ISSN 0022–0302.
El-Deeb, W, Elsohaby, I, Fayez, M, Mkrtchyan, H, El-Etriby, D, ElGioushy, M. 2020. Use of procalcitonin, neopterin, haptoglobin, serum amyloid A and proinflammatory cytokines in diagnosis and prognosis of bovine respiratory disease in feedlot calves under field conditions, Acta Trop. 204:105336.
El-Deeb, W, Fayez, M, Alhumam, N, Elsohaby, I, Quadri, SA, Mkrtchyan, H, 2021. The effect of staphylococcal mastitis including resistant strains on serum procalcitonin, neopterin, acute phase response and stress biomarkers in Holstein dairy cows. Peer J 9:e11511.
El-Deeb, WE, Buczinski, S, 2015. The diagnostic and prognostic importance of oxidative stress biomarkers and acute phase proteins in urinary tract infection (UTI) in camels. Peer J 3:e1363.
El-Deeb, WM, 2013. Clinicobiochemical investigations of gangrenous mastitis in does: immunological responses and oxidative stress biomarkers. J Zhejiang Univ-Sci B (Biomed & Biotechnol) 14:33–39.
El-Deeb, WM, Elmoslemany, A, 2016. The diagnostic accuracy of acute phase proteins and proinflammatory cytokines in sheep with pneumonic pasteurellosis. Peer J 4, 2161. eCollection 2016.
El-Deeb, WM, Ghoneim, I, Fayez, M, Elsohaby, I, Alhaider, A, ElGioushy, M, 2019. Acute phase proteins, proinflammatory cytokines and oxidative stress biomarkers in sheep, goats and she-camels with Coxiella burnetii infection induced abortion, Comp. Immunol. Microbiol. Infect. Dis. 67:101352.
Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med. 1999;340:448–54.
Galvao, K. N., Greco, L. F., Vilela, J. M., Sa Filho, M. F., & Santos, J. E. P. (2009). Effect of intrauterine infusion of ceftiofur on uterine health and fertility in dairy cows. Jounral of Dairy Science, 92, 1532–1542.
Gardner IA, Greiner M. Receiver-operating characteristic curves and likelihood ratios: improvements over traditional methods for the evaluation and application of veterinary clinical pathology tests. Vet. Clin. Path. 2006;35:8–17.
Heegaard, P.M.; Godson, D.L., Toussaint, M.J., TjØrnehØj, K., Larsen, L.E., Viuff, B. & RØnsholt, L. (2000). The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus. Veterinary Immunology and Immunopathology Vol. 77, No. 1–2, (November 2000), pp. 151–159, ISSN 0165–2427.
Hirvonen, J., Pyörälä, S. 1998. Acute-phase response in dairy cows with surgically-treated abdominal disorders. Vet J 1998 Jan;155(1):53–61. doi: 10.1016/s1090.
Hornish, R.E., & Kotarski, S.F. (2002). Cephalosporins in veterinary medicine – Ceftiofur use in food animals. Curr. Top. Med. Chem. 2:717–731.
Jain S, Gautam V, Naseem S. Acute-phase proteins: As diagnostic tool. J Pharm Bioallied Sci. 2011;3(1):118–27.
Kak G, Raza M, Tiwari BK. 2018. Interferon-gamma (IFN-γ): Exploring its implications in infectious diseases. 20018; Bio. Mol. Concepts 2018;9:64–79.
Kim H, GuKang H, KwanJeong J, YoungHur T, HunJung Y. Inflammatory cytokine concentrations in uterine flush and serum samples from dairy cows with clinical or subclinical endometritis. Theriogenology, 2014;82(3):427–32.
Klasing KC, Laurin DE, Peng RK, Fry DM: Immunologically mediated growth depression in chicks: Influence of feed intake, corticosterone and interleukin-1. J Nutr 1987, 117:1629–1637.
Laura L. Healy, James G. Cronin & I. Martin Sheldon. 2015. Endometrial cells sense and react to tissue damage during infection of the bovine endometrium via interleukin 1. SCIENTIFIC REPORTS | 4: 7060 | DOI: 10.1038/srep07060.
Leung, S. T. et al. The effects of lipopolysaccharide and interleukins-1alpha, -2 and – 6 on oxytocin receptor expression and prostaglandin production in bovine endometrium. J. Endocrinol. 168, 497–508 (2001).
Li C, Corraliza I, Langhorne J. A defect in interleukin-10 leads to enhanced malarial disease in Plasmodium chabaudi chabaudi infection in mice. Infect. Immun. 1999;67: 4435–42.
Lu D, Song H, Shi G. Anti-TNF-α treatment for pelvic pain associated with endometriosis. Cochrane Database Syst Rev. 2013;3:CD008088.
Maden M, Ozturk AS, Bulbul A, Avci A, Yazar E. Acute-phase proteins, oxidative stress, and enzyme activities of blood serum and peritoneal fluid in cattle with abomasal displacement. J Vet Intern Med. 2012;26:1470–75.
Meade, K.G., O'Gorman, G.M., Hill, E.W., Narciandi, F., Agaba, M., Kemps S.J., O'Farrellys C., MacHughs D.E (2009). Divergent antimicrobial peptide (AMP) and acute phase protein (APP) responses to Trypanosoma congolense infection in trypanotolerant and trypanosusceptible cattle. Mol Immunol. 2009 Dec;47(2–3):196–204.
Medcalf RL 2007. Fibrinolysis, inflammation and regulation of the plasminogen activating system. Journal of Thrombosis and Haemostasis 5 (Suppl. 1), 132–142. 0233(98)80036–1.
Moore KW, Malefyt DW, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 2001;19:683–765
Murata, H., N. Shimada & M. Yoshioka, 2004. Current research on acute phase proteins in veterinary diagnosis: an overview. The Veterinary Journal, 168, 28–40.
Nasreldin N, Ali FAZ, Abd-Elhafeez HH, Hassan M, El-Zeftawy M, Senosy W. Characterization of immunological, biochemical and inflammatory response of clinical and subclinical endometritis in ewes in the subtropics. Anim Reprod Sci. 2020;219:106541.
Okuda K, Sakumoto R, Okamoto N, Acosta TJ, Abe H, Okada H, Sinowatz F, Skarzynski DJ. Cellular localization of genes and proteins for tumor necrosis factor-α (TNF), TNF receptor types I and II in bovine endometrium. Mol Cell Endocrinol. 2010; 330:41–48.
Opal SM and DePalo VA. Anti-inflammatory cytokines. Chest. 2000;117(4):1162–72.
Petersen HH, Nielsen JP, Heegaard PMH. Application of acute phase protein measurements in veterinary clinical chemistry. Vet Res. 2004; 35:163–87.
Plata-Salaman CR, Borkoski JP: Chemokines/intercrines and central regulation of feeding. Am J Physiol 1994, 266:R1711–R 1715.
Raynes, J. D., 1994. The acute phase response. Biochemical Society Transactions, 22, 69–74.
Richards C, Gauldie J, Baumann H: Cytokine control of acute phase protein expression. Eur Cytokine Netw 1991, 2:89–98.
Rider P, Carmi Y, Voronov E, Apte RN. Interleukin-1α. Semin Immunol. 2013; 25 (6):430–38.
Rubel, C., Fernàndez, G., Dran, G., Bompadre, M., Isturiz, M., and Palermo, M. 2001. Fibrinogen Promotes Neutrophil Activation and Delays Apoptosis. J Immunol; 166:2002–2010.
Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology 2006; 65:1516–30.
Sheldon IM, Noakes DE, Rycroft A, Dobson H, 2001: Acute phase protein response to postpartum uterine bacterial contamination in cattle. Vet Rec 148, 172–175.
Sheldon M, Cronin J, Goetze L, Donofrio G, Schuberth HJ, 2009: Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle. Biol Reprod 81, 1026–1032.
Singh J, Murray RD, Mshelia G, Woldehiwet Z, 2008: The immune status of the bovine uterus during the peripartum period. Vet J 175, 301–309.
Tache´ Y, Saperas E: Central actions on interleukin 1 on gastrointestinal function. In Neurobiology of Cytokines, Part B. Edited by De Souza EB. New York: Academic Press; 1993:169–184.
Tanikawa, M. et al. Regulation of prostaglandin synthesis by interleukin-1alpha in bovine endometrium during the estrous cycle. Prostaglandins Other Lipid Mediat. 78, 279–290 (2005).
Thomas, J. S., 2000. Overview of plasma proteins. In: Schalm’s Veterinary Hematology, 5thedn, eds B. F. Feldman, J. G. Zinkl & N. C. Jain, Lippincott Williams, Wilkins, Philadelphia, pp. 891–898.
Tothova CS, Nagy O, Seidel H, Konvicna J, Farkasova Z, Kovac G, 2008: Acute phase proteins and variables of protein metabolism in dairy cows during the preand postpartal period. Acta Vet Brno 77, 51–57.
Tothova CS, Nagy O, Seidel H, Kovac G. Acute phase proteins and their use in the diagnosis of diseases in ruminants: a reviw. Vet Med-Czech. 2014;59:163–80.
Turner ML, Healey GD, Sheldon IM, 2012: Immunity and inflammation in the uterus. Reprod Domest Anim 47, 402–409.
Wernerry U and Kumar BN. Reproductive disorders in dromedary camels due to infectious causes and its treatment. J. Cam. Prac. Res. 1994;1: 85–7.
Zhang JM and An J. Cytokines, Inflammation and Pain. Int Anesthesiol Clin. 2007; 45(2): 27–37

Table 1: Correlation matrix among acute phase proteins and inflammatory cytokines parameters in she-camel with clinical endometritis.

Variables	HP	SAA	Fg	IFN-γ	TNF-α	IL-1α	IL-1β	IL-10	IL-6
HP	1.00
SAA	0.63	1.00
Fg	0.38	0.55	1.00
IFN-γ	0.73	0.67	0.47	1.00
TNF-α	0.63	0.50	0.25	0.66	1.00
IL-1α	0.54	0.49	0.50	0.65	0.45	1.00
IL-1β	0.53	0.61	0.52	0.63	0.50	0.63	1.00
IL-10	0.50	0.57	0.50	0.62	0.52	0.64	0.60	1.00
IL-6	0.58	0.63	0.62	0.68	0.62	0.65	0.73	0.58	1.00

Haptoglobin (HP), serum amyloid A (SAA), fibrinogen (Fg), interferon gamma (IFN- γ), tumor necrosis alpha (TNF-α), interleukin 1-aplha (IL-1α), interleukin 1-beta (IL-1 β), interleukin 10 (IL-10) and interleukin 6 (IL-6)

Table 2: Diagnostic test characteristics of acute phase proteins and inflammatory cytokines parameters in camels with clinical endometritis.

Parameters	Threshold	Accuracy	AUC	Std. Error	90% confidence interval	P value
HP (g/L)	≥ 0.82	0.87	0.95	0.022	0.92 to 0.99	< 0.0001
SAA (µg/mL)	≥ 12.75	0.92	0.96	0.023	0.92 to 1.00	< 0.0001
Fg (g/L)	≥ 3.85	0.77	0.83	0.053	0.74 to 0.92	< 0.0001
IFN-γ (pg/mL)	≥ 47.30	0.97	0.99	0.008	0.97 to 1.00	< 0.0001
TNF-α (pg/mL)	≥ 128.8	0.95	0.96	0.024	0.92 to 1.00	< 0.0001
IL-1α (pg/mL)	≥ 102.6	0.87	0.91	0.038	0.84 to 0.97	< 0.0001
IL-1β (pg/mL)	≥ 57.60	0.95	0.96	0.025	0.91 to 1.00	< 0.0001
IL-10 (pg/mL)	≥ 91.78	0.95	0.96	0.023	0.92 to 1.00	< 0.0001
IL-6 (pg/mL)	≥ 115.80	1.00	1.00	0.0	1.00 to 1.00	< 0.0001

Table 3. Uterine bacterial isolates from the camel with clinical endometritis

Isolates	She camels (n = 40) n %
Streptococcus pyogenes	5	12.5
Staphylococcus aureus	4	10.0
A. pyogenes	5	12.5
C. pyogenes	8	20.0
E. coli	4	10.0
Salmonella sp.	4	10.0
Proteus sp.	1	2.50
Klebsiella sp.	2	5.00
A. pyogenes + Proteus sp.	2	5.00
Proteus sp.+ E. coli	3	7.50
A. pyogenes + E. coli	1	2.50
Streptococcus pyogenes + E. coli	1	2.50

Download PDF

Editorial decision: Major revision with re-assessment
22 Aug, 2022
Reviewers agreed at journal
02 Aug, 2022
Reviews received at journal
14 May, 2022
Reviewers invited by journal
07 May, 2022
Editor assigned by journal
28 Apr, 2022
First submitted to journal
27 Mar, 2022

You are reading this latest preprint version

Research on acute phase proteins and inflammatory cytokines biomarkers in dromedary camels (camelus dromedarius) with clinical endometritis

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Status:

Version 1