Acute Phase Response In Feedlot Calves With Respiratory Disease
Introduction
Respiratory diseases is considered as one of the most serious problems that causes great losses in calves. Multiple pathogens are identified as causative agents to such diseases condition. Mannheimia haemolytica, Pasteurella multocida, Mycoplasma spp., Histophilus somni (H. somni), and Arcanobacterium (A.) pyogenes are known as bacterial causative agents of BRD. Fever, septicemia, hypoxia, secretion of acute phase proteins (APPs) from the hepatic cells, endotoxin & exotoxin production, lung abscesses, and vasculitis may occur in BRD cases and could lead to myocardial injury. Of note, H. somni did not reported yet as causative agent to BRD in the Kingdom of Saudi Arabia. Taken together, the sequencing analysis of these bacteria isolated from calves reared in the Kingdom of Saudi Arabia didn't demonstrated yet. Of note, H. somni did not reported yet as causative agent to BRD in the Kingdom of Saudi Arabia. Taken together, the sequencing analysis of these bacteria isolated from calves reared in the Kingdom of Saudi Arabia didn't demonstrated yet.Blood biomarkers commonly used in determining the type of the ongoing inflammation and perceiving the body response to the treatment in human medicine. Conversely, in animal medicine a very little number of parameters are used routinely and could be useful in determining the inflammation or evaluating disease outcome.
Recently, APPs are frequently used as non-specific & sensitive markers of inflammation and/or infection. APPs produced in the liver and secreted into blood stream in response to the different pathogens or inflammatory condition. Interestingly, APPs are used in the veterinary field for diagnosis, prognosis of the diseases and evaluation of the treatment efficacy. The release of APPs into the blood varies according to the disease severity and the type of causative agents. In bacterial infection, the acute phase response (APR) is markedly increased, while it is less pronounced during viral infection. Haptoglobin (Hp) and serum amyloid A (SAA) considered the major APPs that studied in bovine species. Hp is a α2-globulin, which has bacteriostatic effect through its ability to bind free hemoglobin. SAA mediates phagocytic cells migration to the infection site and acting as chemoattractant. Higher sreum levels of APPs in cattle is associated with many inflammatory processes as enteritis, endocarditis, urinary tract infection, pneumonia or peritonitis. Importantly, cytokines, which act as messengers between the hepatocytes and infection site, induce the synthesis of APPs and induction of APR.
Production of interleukin (IL) and TNF-α from the macrophage or monocytes in response to infection, induce the synthesize and secretion of APPs from the liver. To date, the routine clinical diagnosis of respiratory diseases in calves become difficult and not determines the causative agents. Moreover, detection of antibodies in the calf serum is difficult in the commencement of the infection and has low sensitivity. However, the maximum production of APPs is estimated within 24-28 h after infection. Thus, determination the levels of APPs & pro-inflammatory cytokines in the animal blood may be considered as a potential tool for BRD diagnosis. Procalcitonin (PCT) which produced in thyroid C cells, is an acute phase protein and a precursor of calcitonin hormone which is responsible for the calcium homeostasis. PCT could be also released from lung and bowel, as well, in cases of sepsis. According to previous studies, PCT is considered as a measurable laboratory marker in inflammatory response in bacterial , fungal, and parasitic infections as it increase rapidly in a very short time after production of pro-inflammatory cytokines (TNF-α, IL-6 and IL-8). It is recognized PCT could increase as one hundred times more than normal serum levels in cases of septicemias.
The data about the diagnostic value of PCT is controversial. While some studies reported that PCT is more reliable than CRP for the diagnosis of neonatal sepsis, others did not find any advantage of PCT over CRP. Thus, the aim of this study was to evaluate PCT as diagnostic and prognostic markers for feedlot calves with BRD.Neopterin (NPT) is a biological biomarker of cell-mediated immunity with low molecular weight that produced by active monocyte/macrophage. IFN-γ is a potential NPT stimulator and it demonstrates the increase in NPT levels and the existence of IFN-γ in body fluids.
The levels of biological markers like IFN-γ, TNF-α, IL-8, PCT and NPT are revealing the results of growing immune response during infections with different pathogens and their levels could be used in perceiving the prognosis in infections.
Notably, no study is correlated yet the serum levels of PCT and NPT in feedlot calves in response to infection with Mannheimia haemolytica and Histophilus somini. The results obtained from this study is expected to help in better understanding the complex mechanism of PCT, NPT, haptoglobin (HP), serum amyloid A (SAA) and cytokines productions in feedlot calves with respiratory diseases under field condition. Therefore, in this investigation, we determine the diagnostic and prognostic accuracy of serum PCT, NPT, HP, SAA and pro-inflammatory cytokines (IL-1β, IFN- ϒ, IL-8, TNF-α) in feedlot calves naturally infected with M. haemolytica and/or H. somini.Material and methodsStudy design:This investigation was conducted on 840 feedlot calves aged between 4-12 month and reared in private farm in Al-Kharg region, Saudi Arabia during 2016 in response to the complaint of farm owner for the presence of respiratory manifestation and deaths among animals. According to the farm reports, all animals were previously vaccinated against viruses causing BRD including, Infectious Bovine Rhinotracheitis (IBR), Bovine Viral Diarrhea (BVD), Bovine Respiratory Syncytial Virus (BRSV), and Parainfluenza Type-3 Virus (PI-3).All animals were clinically examined for signs of bovine respiratory diseases (BRD) (fever, nasal discharge, ocular discharge, congested mucous membrane, abnormal respiratory sounds, depression, decreased appetite, and dyspnea). Nasopharyngeal swabs and blood samples were collected from animals showed high temperature with one or more of the other BRD sings (n=250) for bacteriological and virological investigations.
Animals that exhibited positive bacteriological examination for Histophilus somniand/or Mannheimia haemolytica (n= 69) were received treatment with one antibiotic (Tula"Draxxin®", n=26 cases, FFC "Nuflor®", n=19, Tm "Pulmotil®", n=13, or CEF "Excenel®",n=11) and anti-inflammatory (FM, n=43 or FBZ, n= 26).
Twenty animals showed no response to treatment were necropsied and pharyngeal swabs, tracheobronchial lavage, heart-blood and lung samples were collected for bacteriological and virological investigation.The regimen and doses of treatment for each medicament was conducted according to the manufacture recommendations.Blood samples were taken from BRD cases before treatment and 7- days post treatment. The separated serum samples were stored in Eppendorf tubes at -80 °C for estimation of different biochemical variables including serum amyloid A (SAA), haptoglobin (HP) and Proinflammatory cytokines (IL-1β, IFN- ϒ, IL-8, TNF-α).
Bacteriological culture:The external surface of lung specimens was seared by hot spatula and a parallel incision was made, then after, the deep tissues were swabbed. Nasopharyngeal swabs and lung specimens were streaked on each of brain heart infusion agar (Difco) supplemented with (5% sheep blood agar, 0.5% yeast extract (Difco) and 5 mg/mL of bacitracin) and Colombia blood agar supplemented with 5% sheep blood and selective antibiotics described by (Slee and Stephens 1985) . All agar plates were incubated at 37 °C for 24–48 h under 5% CO2 enriched environment. Colonies displaying morphology indicative of H. somni and M. haemolytica were permissively identified using the VITEK 2 compact, BioMérieux, France.
RNA and DNA extraction: Total DNA was extracted from purified bacterial cultures. Both RNA and DNA were extracted from nasopharyngeal swabs and lung tissues as described previously (Klima et al. 2014), using RNeasy Mini Kit and QIAamp DNA mini kit respectively (Qiagen SA, Courtaboeuf, France) according to the manufacturer’s instructions.16S rRNA amplification and sequencing:The universal bacterial primers 27F (5′-AGRGTTTGATCMTGGCTCAG) and 1492R (5′-GGTTACCTTGTTACGACTT), (Lane et al 1991), were used for amplification of 1400bp of bacterial 16S ribosomal RNA. PCR reaction and amplification was performed following the methods described by Holman et al. (2015) using I cycler PCR machine (BIO-RAD, California, USA).
The amplified PCR products were visualized in 0.8% agarose gel stained with ethidium bromide using ultraviolet gel documentation system (BIO-RAD, USA).For sequencing, the PCR products were purified with QIAquick PCR purification kit (Qiagen SA, Courtaboeuf, France) according to the manufacturer’s instructions. Genetic analyzer 3500, (Applied Biosystems) was used for sequencing. 16S rRNA sequences were subjected to NCBI Basic Local Alignment Search Tool (BLAST) analysis.
Phylogenetic analysis: In the phylogenetic study, two approaches were followed to analyze the phylogenetic relations of the obtained Histophilus and Mannheimia sequences. In the first approach, the obtained sequences were aligned with representative strains from family Pasteurellaceae to figure out the possible position with family outliers. The second approach was adopted to discover the most similar sequence with high identity to the recovered sequences regardless family restrictions. For this purpose the obtained sequences were used to blast nonredundant databases of microbial nucleotides. The top most relevant 100 sequences with the highest identity was retrieved. This set will help to find the most common ancestor for the retrieved sequences in this work. ClustalW alignment was generated by the Geneious software version 11. The phylogenetic tree was built on neighbor-joining method. Neighbor-Joining bootstrapping for up to 100 replications was used to assess the reproducibility of the generated tree nodes. The trees were visualized and annotated by genedoc and Geneious software. Similarity matrices were generated after comparative multiple pairwise comparison of sequences.PCR for detection of Viral and bacterial pathogen.
DNA and RNA extracted from nasopharyngeal swabs and (pharyngeal swabs, tracheobronchial lavage, heart-blood and lung samples) from necropsied animals were amplified for detection of M haemolytica and H somni and BVDV, BHV-1, BRSV, PI3V according the methods after (Klima et al 2014). HotStarTaq® Plus Master Mix Kit (QIAGEN, USA) was used for PCR reaction according to the manufacturer’s instructions. PCR conditions and primers were used according to the methods of Biochemical analysis of selected biomarkersThe separated serum samples (before and after treatment) were stored in Eppendorf tubes at -80°C for estimation of different biochemical variables.Among the blood samples, PCT, IL-1β, IFN-γ, IL-8, TNF-α, levels were determined through the sandwich enzyme immunoassay method. The levels of NPT levels were determined in serum samples through competitive inhibition enzyme immunoassay method and through commercial kits and in accordance with the kit procedures and by using the ELISA deviceBlood samples were taken from BRD cases before treatment and 7- days post treatment.Proinflammatory cytokines (IL-1β, IFN-ϒ, IL-8, TNF-α) were analyzed using ELISA test kits (CUSABIO). Analysis of serum Hp levels and SAA in both groups was carried out using ELISA kits (Phase SAA kit, Tridelta Ltd., Ireland) according to the manufacturer’s instructions.Statistical analysisVariations in studied biological biomarkers in the feedlot calves were compared using Wilcoxon Mann-Whitney test analysis at P-value < 0*05. Selection of different cutoff points that optimize sensitivity (Se) and specificity (Sp) for each of PCT, NPT, Hp, SAA and proinflammatory cytokines were detected using receiver operating characteristics (ROC) analyses. The ROC curves were plotted as Se versus 1–Sp (false positive rate) for all possible cut-off points for PCT, NPT, Hp, SAA, IL-1β, IFN-γ and IL-8. The differences in the AUC for the PCT, NPT, Hp, SAA, IL-1β, TNF-α, IFN-γ and IL-8 were evaluated using a non-parametric method that describes the correlation resulting from using the same sample for both tests. The complete data analyses were conceded by using Stata version 13 (Stata Corp, College Station TX, USA).
Results
Clinical appearance Sixty nine feedlot calves with Mannheimia haemolytica and/or Histophilus somni were exhibited different clinical signs of BRD as shown in table 2 .Feedlot Calves that were apparently healthy with no isolated pathogenic bacteria or virus (n = 20) were served as negative control. Abnormal lung sounds and cough were detected in pneumonic feedlot calves with a significant elevation (P < 0.05) in body temperature, respiratory rate and heart rate in comparison with healthy controls. The clinical findings of diseased calves were assessed 7- days after treatment with different antibacterial agents combined with non- steroidal anti-inflammatory drugs. According to the response to treatment, the diseased calves were further categorized into responsive (n= 49) and non-responsive group (n= 20) to the treatment trials. Within the first 7-days after initiation of the therapy, the mean body temperature, respiratory rate, heart rate and abnormal lung sounds were returned to the normal levels in 49 calves. However, the clinical findings (fever, cough, nasal discharge, abnormal lung sounds) were still detected in 20 calves.
Bacterial and viral pathogen detection: Overall, H. somni and M. haemolytica were isolated from 69 nasopharyngeal swabs. H. somni was isolated from 49 cases and M. haemolytica from 58 cases. Coexistence of both bacteria was found in 38 cases. The 16S rRNA gene sequences from each isolate was subjected to BLAST analysis and representative sequences were submitted to GenBank with accession no MG725885, MG725884.1, MG725883, MG725828, MG725827,MG725826.1,MG725726.All samples were negative for BVDV, BHV-1, BRSV, and PI3V by PCR examination.PCT, NP, HP, SAA, IL-1β, IL-8, TNF-α, IF-γ in sera Significant elevations (P < 0.05) were detected in PCT, NP and all assessed pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, IF-γ), in diseased feedlot calves in comparison with the healthy control group (Table 1). Table 2 showed the diagnostic accuracy of selected biomarkers in feedlots with BRD. The results revealed a high degree of diagnostic accuracy of PCT, NP, IL-1β, IL-8, TNF-α, IF-γ (0.99, 0.98, 0.925, 0.97, 0.96, and 0.94 respectively) at the selected thresholds.Table 3 showed the prognostic accuracy of selected biomarkers in feedlots with BRD. The table declared a high degree of prognostic accuracy of PRC and NP, IL-1β and IF-γ in feedlots with BRD at the selected thresholds. While, at 7- days' post-treatment, significant inhibition (P < 0.05) in the serum levels of all selected biochemical markers were observed in all treated calves (Table 4) toward the levels of control healthy feedlot calves. Interestingly, the serum levels of IL-8 were returned to the normal levels in diseased calves treated with combination therapy of different antibacterial agents and non-steroidal anti-inflammatory agents.
Discussion
To the best of author's knowledge, this is the first report to investigate the serum levels, diagnostic and prognostic accuracy of PCT, NPT in feedlot calves under field condition. Moreover, this study address the prognostic importance of PCT, NPT, HP, SAA and proinflammatory cytokines in feedlots for the first time. Procalcitonin (PCT) which produced in thyroid C cells, is an acute phase protein and a precursor of calcitonin hormone which is responsible for calcium homeostasis. PCT could be also released from lung and bowel, as well, in cases of sepsis. According to previous studies, PCT is considered as a measurable laboratory marker in inflammatory response in bacterial , fungal, and parasitic infections as it increase rapidly in a very short time after production of pro-inflammatory cytokines (TNF-α, IL-6 and IL-8). In this study, PCT showed a significant increase in their levels in feedlot calves infected with M. haemolytica and/or H. somni when compared with control calves. It was stated that, serum levels of PCT increased rapidly in bacterial diseases shortly after the release of certain pro-inflammatory mediators (TNF-α, IL-6 and IL-8). It was reported by various researchers that PCT could increase as one hundred times more than normal serum levels in cases of septicemias.
Interestingly, It was previously detected that PCT levels was under 0.1 ng/ml in healthy persons, >0.5 ng/m1 in viral infections and 1.5 ng/ml as highest. Furthermore, in sever bacterial infections this level may increase to five more times at least, that it could go beyond 10ng/ml (Baylan et al., 2002; Tünger, 1998). NPT is existing in very small amounts in different body fluids; it detected before the presence of specific circulating antibodies, and often precedes clinical signs of the disease. It is secreted from monocyte/macrophage. The NPT assays in veterinary medicine is scarce and has so far only been investigated by a few authors. They measured NPT in cattle, pigs, llamas, dogs, cats, rabbit and rats. In this study, NPT increased considerably in feedlot calves infected with M. haemolytica and/or H. somni when compared with controls. The higher serum levels of NPT attributed to the activation of the cellular immune response. Shaw (1991) and Facer (1995) in viral, bacterial and parasitic disease previously reported comparable observations. Recently, Ercan et al. (2016) reported similar findings in calves with septicemic colibacillosis. High comparable levels of NPT reported previously in different inflammatory conditions and certain malignancies.
NPT is biochemically inert and stable as its half‐life in the body is merely due to renal excretion, consequently detection of NPT has numerous advantages compared to cytokines which has a short half-life and very liable for fast degradation.In this study, the Hp levels elevated 21 times in feedlot calves with BRD indicating a strong immune response to the M. haemolytica and/or H. somni infection. Elevated serum level of Hp could be convinced by cell injury following infection and/or inflammation. Hp considered as potent bacteriostatic protein against different bacterial pathogens by binding free hemoglobin, so depriving bacteria from iron required for their growth. These findings are comparable with those reported by many authors in calves with natural respiratory infections and in calves experimentally infected withP. multocida. Similar results detected in pneumonic sheep and by El-Bahr & El-Deeb, (2013) in buffalo calves with bacterial bronchopneumonia. Conversely, some authors (Wittum et al., 1996 and Young et al., 1996) previously reported limited association between respiratory diseases and Hp levels in feedlots. Our study also declared higher levels of SAA approximately four folds in feedlots with BRD compared to control feedlots showing a moderate acute phase response. The SAA response could be attributed to its significant role in modulating immune defense of animals during infection and/or tissue injury. Moreover, SAA alters cholesterol metabolism during different inflammatory problems. In addition, it was stated that SAA has the ability to inhibits Gram-negative bacteria, probably to simplify the process of phagocytosis of pathogenic bacteria. Likewise, some authors reported that SAA was noticeably elevated in pneumonic calves. The selected pro-inflammatory cytokines in our study (IL-1β, IL-8, TNF-α, IF-γ), showed a higher serum values in feedlot calves with M. haemolytica and/or H. somni infection than that of control. These results revealed that such infection was associated with a solid APR and pathological changes in infected feedlots were correlated with the levels of pro-inflammatory cytokines. In addition, elevation of these cytokines may be an indicator of inflammatory process related to BRD as marked by high diagnostic sensitivity and specificity.
The current results provides a strong evidence that support the important role of IL-1β, IL-8, TNF-α, IF-γ in BRD pathogenesis in feedlot calves.These findings are in consistent with those reported in different animals with bacterial infection. Moreover, the expression of TNF- α and IL1-β were elevated in the lung lesions and respiratory passages of infected calves with Mannheimia haemolytica (Malazdrewich et al., 2001). The area under the ROC curve for the tested biological markers (PCT, NPT, Hp, SAA, TNF-α, IL-1β, IL-8 andIFN-γ) showed a high degree of accuracy that support the clinical diagnosis of infected feedlots with M. haemolytica and/or H. somni. The application of APPs and proinflammatory cytokines in diagnosis of infection has been reported by many authors.
The area under the ROC curve for the tested PCT, NPT, IL-1β, IL-8 and IFN-γ showed a high degree of accuracy in prognosis of infected feedlots with M. haemolytica and/or H. somni. Many authors have documented the application of PCT, NPT, IL-1β, IL-8 and IFN-γ in in the prediction of recovery in both human and animals.
Based on our results, PCT, NPT, IL-1β, IL-8 and IFN-γ could be used to monitor the response of infected feedlots (with M. haemolytica and/or H. somni) to the treatment in a real-time manner.In this study, the ability of PRC, NP, HP, SAA and proinflammatory cytokines to discriminate between BRD cases and healthy feedlots was assessed using ROC analysis. Under the conditions of this investigation, PRC, NP, IL-8, TNF-α and IFN- ϒ showed a high degree of discrimination between BRD cases and control feedlots (AUC > 0.9) according to guidelines stated by Swets (1988). On the other side HP, SAA, IL-1β showed a moderate degree of discrimination between BRD cases and control healthy calves (AUC> 0.8). Selection of the proper cut-off point for each examined biomarkers was based on optimizing sensitivity and specificity which lead to the best overall correct classification.In this study, the ability of PRC, NP, HP, SAA and proinflammatory cytokines to assess the prognosis of BRD cases was evaluated using ROC analysis. Under the conditions of this investigation, PRC, NP, IL1-β and IFN- ϒ showed a high degree of discrimination between responsive and non-responsive cases to the treatment protocol (AUC= 1) according to guidelines stated by Swets (1988). On the other side HP, SAA, IL-1β showed a moderate degree of discrimination between BRD cases and control healthy calves (AUC= 1).
