MOLECULAR, BACTERIOLOGICAL AND CLINICAL PATHOLOGICAL STUDIES ON PNEUMONIC CALVES WITH SPECIAL REFERENCE TO ANTIBIOTIC RESISTANCE GENES

Assiut University web-site: www.aun.edu.eg

MOLECULAR, BACTERIOLOGICAL AND CLINICAL PATHOLOGICAL STUDIES ON PNEUMONIC CALVES WITH SPECIAL REFERENCE TO ANTIBIOTIC

RESISTANCE GENES

HALA A. ABD EL-HAMED ¹ and GHADA A. IBRAHIM ²

¹ Clinicalpathology, Animal Health Research Institute, Egypt

² Bacteriology Units Ismailia, Provincial Laboratories, Animal Health Research Institute, Egypt

Received: 28 September 2017;       Accepted: 31 October 2017

INTRODUCTION

Respiratory infections are responsible for 37-52% losses in cattle. Unfortunately, Calves experienced pneumonia at early age might have severe depression in the future in the production capabilities causing severe economic costs (Sayed and Zaitoun, 2009 and Griffin et al., 2010).

Bacteria and viruses in combination with stress factors are the key in triggering acute respiratory infections usually bacteria act as the second invaders to worsen the ill-animal's condition (Yousef et al., 2013). The most bacterial causes include: Staphelococcus aureus, Staphelococcus Pneumonae, Escherichia coli, pseudomonas spp., Klebsiella spp, Mycoplasma haemolytica and Pasterella multocida.

Corresponding author: Dr. GHADA A. IBRAHIM

E-mail address: g_abdelaall@yahoo.com,

Present address: Bacteriology Units Ismailia, Provincial Laboratories, Animal Health Research Institute, Egypt.

The emergence of antibiotic resistant pathogenic bacteria has become a serious problem (French, 2005). The presence of resistant bacteria poses a risk to humans as they may act as resistance reservoirs, contributing to the maintenance and spread of antibiotic resistance genes (Goni-Urizza et al., 2000).

Clinical signs of respiratory disease in a group of calves are discordant. They includes: elevated rectal temperature, frequent coughing, muco-purulent nasal/ocular discharge, lethargy, loss of appetite and frequent lying down, exaggerated vesicular sound and moist rales with frictional sound may be heard (NADIS, 2014).

Almujalli et al. (2015) indicated that leukocytosis with neutrophilia was a hematological finding associated with pneumonia in calves. Moreover, there was a significant elevation in the levels of ALT, AST, ALP and AGP with significant decrease in levels of total protein, albumin in pneumonic calves in comparison with control calves. Saleh and Allam, (2014) revealed that the serum enzyme activity of ALT and AST were elevated (P<0.05) in sheep with pneumonia. Furthermore, their results added that there also a significant (P<0.05) decrease in serum concentration of albumin in association with a significant (P<0.05) increase in serum levels of gamma globulins (γ-Glob.) in the examined pneumonic sheep. The serum biochemical change of pneumonic goat due to Pasteurlla multocida  indicates significantly lower in Ca and Mg levels and increase in the concentration of inorganic phosphorus in comparison with the healthy goat (Sadeghian et al., 2011). A significant association with blood sample collection day and a decrease of Cl, K, and P concentrations and interactions between blood sample collection day and the severity of pneumonia in Ca and Na concentrations in calves after experimentally induced pneumonia, found (Fraser et al., 2014). The available data on the effect of respiratory disease on the mineral status, mainly trace elements, in calves were few.

Therefore, this work aims to investigate the clinical picture, evaluate the hemato-biochemical alterations and pulmonary function tests in pneumonic calves with reference to their microbial causes and their virulence factors. The current status of drugs sensitivity and resistance patterns using PCR technique in the diseased calves is also assessed.

MATERIALS AND METHODS

1. Animals: One-hundred and thirty Friesian calves from different farms belonging to Ismailia Governorate, Egypt, aged 2 to 9 months and their weights ranged from 60-130 kg were clinically examined they classified into 2 groups: The 1^st consisted of 40 apparently healthy calves that didn’t show any diseased condition and didn’t expose to any treatment; they were kept as a control calves. The 2^nd group consisted of 90 calves show respiratory manifestation presenting clinical signs of pneumonia including fever (39.7±0.71 ºC), dyspnea (n=50), mouth breathing, nasal discharge (n=50), coughing (n=50), depression (n=50), weight loss, increased respiratory rate (22.6±2.9 min–1).

2. Clinical examination: All animals were subjected to clinical examination according to Rosenberger et al. (1979). Data concerned with the case history, clinical findings, and medical record for each calf were illustrated in (Table 11).

3. Samples:

a) For bacteriological examination: Nasal swabs and blood smears samples were collected from the examined calves (90 pneumonic and 40 apparently healthy calves) from different farms in Ismailia Governorate in Egypt. Samples were individually collected using sterile swabs with nutrient broth as a transport medium then labeled and transported immediately to the bacteriological laboratory for examination.

b) For haematological examination: Blood samples were collected from both groups and were divided into three parts. The first part was collected on (EDTA) for hemogram. The second part was collected on heparin (20 IU/ ml) for measuring of plasma values of fibrinogen (F) The samples were placed in a bed of crushed ice, taken immediately to the laboratory for analysis. The third part was placed in a plain centrifuge tubes for separation of serum. The serum samples were stored at -20ºC until assayed for the rest biochemical parameters.

4. Hematological studies: The evaluated hematological parameters in this study included estimation of red blood cell count (RBCs), hemoglobin concentration (Hb), packed cell volume (PCV), Total leukocytic count (TLC) and differential leukocytic counts. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated. These parameters were performed according to the routine hematological procedures adopted by Feldman et al. (2000).

5. Biochemical examination: Serum protein was determined according to method illustrated by Doumas et al. (1981). Albumin and globulins were separated by cellulose acetate electrophoresis using Helena system (Helena France) (Batavani et al., 2006). Serum enzymes AST was estimated according to method previously described by Kachmar and Moss (1987), ALT was estimated according to method previously described by Bergmeyer and Harder (1986). Serum creatinine was determined using kits (Bio-labo, France) according to method described by (Young, 1995). Analysis of urea was carried out by using commercial test kits (Vitro Scient, Egypt) according to method described by (Rock et al., 1987). Sodium and potassium values were estimated by using of atomic absorption spectrophotometer (Perkin-Elmer, 1967). Serum concentrations of calcium (Ca, mmol/l), magnesium (Mg, mmol/l) glucose (Glu, mmol/l), phosphorus (P, mmol/l), creatine phosphokinase (CK, μkat/l) and iron (Fe, μmol/l) were analysed by atomic absorption spectrophotometer (A Analyst 100, Perkin Elmer). Plasma fibrinogen was detected by Spectrophotometric method using commercial kits of Boehringer Ingelheim (Germany).

6. Isolation and identification of bacterial causes: All nasal swabs samples were transported on nutrient broth medium, cultivated and aerobically incubated at 37 °C for 24 hours. In 2^nd day, they were streaked on nutrient agar, 10% sheep blood agar and MacConkey's (Oxoid) agar media for each sample for cultivation of both Gram positive and Gram negative bacteria. The cultured plates were incubated aerobically overnight at 37 °C for 24 hours. Pure colonies from the recovered isolates were sub-cultured on selective agar media: Eosin Methylene Blue and Mannitol salt agar with aerobic incubation at 37°c for 24 hours. Microscopic examination of the recovered isolates with Gram stain was done. However, blood smears were stained with Gimsa stain for identification of Pasteurella spp. The suspected colonies were purified and biochemically tested based on the criteria of Quinn et al. (2002). Catalase, Oxidase, indole, motility, Triple Sugar Iron agar slants, citrate utilization, methyl red and urea production. The Identified E. coli isolates were serotyped by commercially available kits using polyvalent and monovalent antisera O and K (Test Sera Enteroclon, Anti –Coli, SIFIN Berlin, Germany) at Animal Health Research Insititute, Serlogy Unit, Dokki, Giza. All the isolates were stored in brain heart infusion broth with 30% glycerol at –70°C until required.

7. Pathogenicity test for Past. Multocida isolates (Buxton, and Fraser, 1977): Three Swiss mice weighting about 15-20 g for each isolate were used. All mice were injected intrapritoneally with 0.1 ml of the cultured bacterial suspension with infectious dose of (1.5x10⁸cfu). Three mice for each isolate were used as a control (which was injected I/P with 0.1 ml of sterile normal saline). All mice were kept under observation and mortality rate was recorded. Re-isolation of the organism from the dead mice was carried out from heart blood and from spleen; liver and lung on 10% sheep blood agar medium as was previously mentioned. The blood films were prepared and stained with Gimsa stain for showing the characteristic features of Past. multocida organisms.

8. Antimicrobial susceptibility test: The susceptibility profiles of (E. coli, S. aureus and pasterella multicoda) isolates from pneumonic calves were performed using disk diffusion technique according to the procedures of (CLSI, 2011). Pure colonies were picked up, cultivated on Muller Hinton broth and incubated at 37˚C for 24h then compared with 0.5% of McFarland tube. Then the bacterial suspension was streaked on Mueller-Hinton agar plates using a dry sterile cotton swab and incubated at 37˚C for 24 h. The inhibition zone diameter of the cultured plates were recorded and measured. The following antibiotics were assayed: penicillin (10 µg), amoxicillin+clavulanic acid (10 µg), gentamicin (120 µg), erythromycin (15 µg), enrofloxacin (5 µg), sulphamethoxazole (25 µg), tetracycline (10 µg), ciprofloxacin (5µg), and norofloxacin (10 µg).

9. Molecular identification of some virulence and resistance genes:

DNA extraction: DNA extraction with modifications of 24 hours incubatedbuffered peptone suspensions of some isolated bacterial strains (E. coli, S. aureus and past. multicoda) according to the manufacturer’s recommendations at RLQP (Reference Laboratory for Veterinary Quality Control on Poultry Production) was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) for screening for some virulence and antibiotic resistant genes with conventional PCR. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Oligonucleotides primers were supplied from metabion (Germany).

PCR amplification: Primers were utilized in a 25 µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an Appliedbiosystem 2720 thermal cycler.

Analysis of the PCR Products: The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the uniplex PCR products were loaded in each gel slot. Generuler 100 bp DNA Ladder (Fermentas, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software (Sambrook et al., 1989). For each PCR experiment, appropriate positive and negative controls were included.

10. Statistical Analysis

Statistical analysis was performed using computer program statistical package for social science (SPSS) Leech et al. (2007). Results were expressed by mean ± SEM and all the comparisons were done by ANOVA method and considered different when p < 0.01.

Table1: Oligonucleotide primer sequences of virulence genes of bacterial isolates.

F: Forward            R: Reverse

Table 2: Oligonucleotide primer sequences of antibiotic resistance genes of isolates.

F: Forward            R: Reverse

Table 3: Cycling conditions and predicted sizes of PCR products for virulence and antibiotic resistance gene.

RESULTS

Bacteriological examination of bacterial pathogens in calves:

The present study recognized that E. coli (34.6%), S. aureus (28.5%) and Pasterella multocida (13.1%) were that most predominant isolated pneumonic bacterial pathogens among 40 apparently healthy and 90 pneumonic calves showing pneumonia and respiratory disorders (Table 4).

Phenotypic and cultural characterization of the recovered isolates: According to morphological and cultural characters, E. coli appear microscopically as Gram negative medium sized bacilli. It grows on macconky's agar medium as pink non-lactose fermenter colonies and gives the characteristic green metallic sheen appearance on EMB agar. Biochemically, E. coli isolates were indole positive, citrate negative, urease negative. Serotyping of the recovered E. coli isolates in this study revealed different serotypes (O143: H4, O1: H2, O63: H5, O157:H7, O158: H10, O119:H4, O86:H9 and O18:H6) (Table 5). In addition, S. aureus isolates were appeared microscopically, as Gram-positive cocci, non-motile, non-spore forming, non-capsulated and usually arranged in grapes like irregular clusters. They were β- heamolytic and change the colour of (MSA) medium from pink to medium yellow due to sugar fermentation and acid production. Biochemical identification showed that they were sugar fermenters, catalase, coagulase, citrate and urease positive while negative for indole and oxidase tests. However, Past.multocida colonies exhibited smooth glistening and translucent on nutrient agar while they were non-hemolytic dewdrop like colonies on 10% sheep blood agar media but didn’t grow on MacConkey agar media. They were Gram negative, coccobacilli and with biochemical identification, Past. Multocida were positive for indole, oxidase and catalase, while negative for citrate and methyl red tests. Concerning to, the results of pathogencity test of Past. multocida isolates in mice; they were highly pathogenic in mice producing acute septicemia and death within 24-48 hours post inoculation (Table 6). Bipolar organisms were clear microscopically in Giemsa stained smears that were prepared from heart blood of dead mice. Past.multocida was re-isolated from all inoculated mice from the blood, liver, spleen and lung samples on 10% Sheep blood agar and examined microscopically with Gimsa stain.

Antimicrobial resistance profile of the tested bacterial isolates: Table (7) indicated that E. coli isolates were of high resistance rates against amoxicillin+clavulanic acid, penicillin, ciprofloxacin and sulphamethoxazole (100%) followed by tetracycline (80%). S. aureus isolates showed high resistance levels to penicillin and norofloxacin (100%) followed by tetracycline (90%). However, Past.multocida isolates exhibited high sensitive rates against most antibiotics were used however, they were resistant totetracycline (41%) followed by enerofloxacin and norfloxacin antibiotics (30% for each).

PCR screening for some specific virulence genes for the recovered isolates: Among ten E. coli examined isolates with conventional PCR in this study; iss and pap C virulence genes were detected in 90% and 60% of the isolates, respectively as shown in (Table 8) and (Fig.1, A &B). The virulence genes of S. aureus (Spa and clfA) genes were found in 100% and 80% of PCR tested isolates, respectively (Fig.1, C&D). In addition, PCR identification of some recovered Past. Multocida isolates with Tox A gene detected clear characteristic bands which were observed at 864 bp of (Fig.1,E) in all isolates. Also, kmt1 virulence gene was detected with PCR in 100% of the isolates (Fig.1,F) indicating their pathogenicity and virulence of Past. Multocida isolates.

Genotypic characterization of antibiotic resistance genes of the tested isolates: In the present study, the phenotypic resistance of E. coli isolates to amoxicillin-clavulanic acid and ciprofloxacin antibiotics could be explained by the presence of bla_TEM and qnrS resistance genes among the twenty examined isolates (100% for each) however, tetracycline resistant gene (tetA) was found in 80% of E. coli isolates as (Table 9). PCR amplifications of the resistance genes yielded the predicted amplicon sizes at 516 and 417 bp and 576 bp (Fig2. G&H&I). According to S. aueus isolates, NorA was exhibited in 100% of the examined isolates (Table 10) that they were phenotypically resistant to norofloxacin however, blaZ gene was detected in 90% of S. aueus PCR examined isolates (Fig2. J&K).

The clinical signs in this study in Table (11) showed that a significant increase in both respiratory and heart rates in calves affected with pneumonia and wheezing with a high pitched breath sound was also detected during thoracic auscultation indicating severe lung injury. According to the hematological results were shown in Table (12), there was a significant (P<0.05) decrease in RBCs count and Hb, PCV, MCV, MCH and MCHC values in the diseased group compared to the control. There was also seen significant (P< 0.05) increase in TLC was seen in the diseased group with significant (P<0.05) increase in the mean values of neutrophilic, eosinophilic and monocytic counts. The mean values of lymphocytic count showed a significant (P< 0.05) decrease in the diseased group while no changes were observed in the count of basophils. Also, the results of serum biochemical changes as shown in Table (13,14&15) clarified that significant (P<0.05) decreases were noticed in serum concentration of albumin and A/G ratio in diseased group compared to the control one but serum values of total proteins and globulins showed a significant increase. Serum concentrations of urea, creatinine and serum enzymatic activities of ALT and AST were significantly (P<0.05) increased in the diseased calves. The mean values of serum levels of P and Na were significantly (P<0.05) lower in the diseased calves.

Table 4: Isolation rate of bacterial species in apparently healthy and pneumonic calves.

Table 5: prevalence of detected serotypes based on total number of E.coli isolates (n=45).

Table 6: Pathogencity test of the isolated Past. Multicoda strains in mice.

Table 7: Antibiotic sensitivity patterns of most isolated bacterial species.

S: sensitive strain                                                                                              R: resistant strain

Table 8: The prevalence of virulence and antibiotic resistance genes among examined isolates.

Table 9: Relation between resistance profiles and genotypic characterization of some virulence and antibiotic resistance genes of E.coli isolates.

Table 10: Relation between resistance profiles and genotypic characterization of some virulence and antibiotic resistance genes of S. aureus isolates.

Table 11: Clinical Findings in pneumonic and apparently healthy calves.

Abbreviation: R.R: Respiratory Rate; H.R: Heart Rate Significant differences in the values between the diseased and control groups were indicated by * Significant P< 0.05

Table 12: Mean values ± SD of blood cell parameters of the pneumonic calves compared to the control healthy group.

Significant differences in the values between the diseased and control groups were indicated by* Significant P< 0.05

Table 13: Mean values ± SD of Changes in serum biochemical parameters of the pneumonic calves compared to the control healthy group.

Significant differences in the values between the diseased and control groups were indicated by* Significant P< 0.05     

Table 14: Mean values ± SD of serum total protein and protein electrophoresis in the pneumonic calves compared to the control group.

Significant differences in the values between the diseased and control groups were indicated by

 * Significant P< 0.05 ** Highly significant P <0.01

Table 15: Electrolyte profile and trace elements status (mean values ± SD) in the tested calves with Pneumonia.

Significant differences in the values between the diseased and control groups were indicated by * Significant  P< 0.05Calcium (Ca, mmol/l), Magnesium (Mg, mmol/l), Sodium (Na, mmol/l), Potassium (K, mmol/l), Fibrinogen (F, g/l ) and Iron (Fe, μmol/l) phosphorus (P (µM/L).

Fig. (1): Shows agarose gel electrophoresis of PCR amplified products of (A&B): iss and papC virulance genes of E.coli isolates, (C&D) spa and clf A virulance genes of S.aureus isolates, (E&F): toxin A and kmt₁  virulance genes of Past.multocida isolates. Lane M: DNA molecular size marker (100 bp), lanes 1-10: The examined isolates except (E&F): lanes 1-5 of Past.multocida isolates, lane (+ve): positive control and lane (-ve): negative control. The size in base pairs (bp) of each PCR product is indicated above the bands.

Fig. (2): Shows agarose gel electrophoresis of PCR amplified products of (G&H&I): tetA, bla_TEM and qnrS antibiotic resistance genes of E.coli isolates, (J&K) norA and blaz antibiotic resistance genes of S.aureus isolates. Lane M: DNA molecular size marker (100 bp), lanes 1-10: The examined isolates, lane (+ve): positive control and lane (-ve): negative control. The size in base pairs (bp) of each PCR product is indicated above the bands.

DISCUSSION

Respiratory diseases constitute the most frequently causes of high morbidity and mortality in calves (Radostits et al, 2007). Generally, they are a complex syndrome that involve stress and environmental factors, bacterial, fungal and viral infections which usually could develop when the immune system of the animal is compromised or if the calf has a concurrent bacterial or viral infection (Shahrour, 2003).

E. coli is a major frequent causative agent of pneumonia in calves (El-Shabrawy, 2005). In addition, S. aureus is a commensal of mucous membranes especially in the respiratory tracts in humans and animals (Carter, 1986) and most commonly isolated from pneumonic calves (Ismail et al., 1993) and also, Past. Multocida is more frequently associated with pneumonia in dairy calves (Bryson, 1985).

Bacteriologically, in this study, the prevalence ratio of isolated pneumonic bacterial pathogens was: E. coli (34.6%), S. aureus (28.5%) and Past. multocida (13.1%). Enany et al. (2012) revealed the predominance of E. coli (36.14%) in 70 nasal swabs of buffloe calves wher as Sayed and Zaitoun (2009) indicated that S. aureus (22.43%) is the predominant isolate among bacterial species from pneumonic calves; meanwhile E. coli and P. multocida were found in lower percentages (18.22%) and (15.89%), respectively. Kassahun Gebremeskel et al. (2017) isolated S. aureus (22.43%) and E.coli (18.22%) but in lower percentages. Also, Seker and Yardimci, (2010) isolated E.coli (16.2 %), S. aureus (16.3%) and Past. Multocida (8.4%) among the 165 Gram positive and Gram negative bacterial isolates from the nasal cavity of buffalo calves. The variation in the isolation rates could be attributed to the change of hygienic, management factors and immune status of the animals (Sedeek and Thabet, 2001).

E. coli serotyping in (Table 5) showed that however, most frequently isolated E. coli strains in this study were extra-intestinal pathogenic E. coli (ExPEC). Smith et al. (2007) confirmed that ExPEC strains possess virulence traits that allow it to invade, colonize, and induce disease in body sites outside of the gastrointestinal tract when they leave the GI tract and infect other parts of the body such as the urinary tract, the blood, or the lungs, illness results causing infections and illness in other extraintestinal locations in human and animals (pneumonia, urinary tract infections, neonatal meningitis). 

The results of pathogencity test of the isolated Past. multocida in mice (Table 6) indicated that they were pathogenic. These results agree with previously mentioned with Enany et al. (2012) and Varte et al. (2014) who reported that all the field isolates of Past. Multocida were found pathogenic for mice and killed the mice inoculated within 6-24 hours post-infection.

Both E.coli and S. aureus isolates developed high resistance levels against different used antimicrobials but Past. multocida exhibited low resistance rates (Table 7). Generally, the resistance to the antimicrobial agents is likely related to their widespread and unreasonable use in the veterinary field. Enany et al. (2012), Ouchriah et al. (2015) and El-Shehedi et al. (2016) confirmed high antimicrobial resistance of the same isolated strains from nasal and lung samples of calves.

The presence of multiple virulence factors increases the virulence potential of bacterial strains. The virulence genes were detected using species specific primer with PCR technique for the recovered isolates. Extra-intestinal pathogenic E. coli virulence potential is attributed to the presence of specialized virulence factors that help the microorganism to cause the disease (Sabarinath et al., 2011). The iss gene has a vital role in the pathogenicity of E. coli and could be a potential target for developing novel therapeutics and prevention strategies. Moreover, pap C gene, the main functional gene of P pilus, is involved in adhesion of pathogenic E. coli to the host cells. The prevalence of issand pap C virulence genes of E. coli isolates were 80% and 60% of the isolates. Higher prevalence (100% and 81.8%) of iss and pap C among E. coli isolates were recorded by Ammar et al. (2015).

S. aureus encodes many proteins that act as virulence factors. Among these virulence factors: SpA and ClfA  are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses (Stutz et al., 2011). Moreover, the spa gene is an important virulence factor of S. aureus because it is useful for the identification and typing of methicillin resistant S. aureus (MRSA) (Dag Harmsen et al., 2017). In the current study, all the isolated strains of S. aureus were MRSA that impair the opsonisation process by serum complement and phagocytosis by polymorphonuclear leukocytes. Clumping factor A (Clf A gene) was detected in 80% of the isolates. Clf A gene is one of the essential adhesion and virulence factors for S. aureus (Heilmann, 2011).

Toxigenic and non-toxigenic Past.multocida isolates couldn’t be differentiated by morphology or standard biochemical reactions. PCR is accurate, rapid and specific for detection of toxigenic Past. multocida (Carol et al., 1996). Some virulence factors such as dermonecrotoxin are essential for the virulence and pathogencity of Past. Multocida strains (Tox A gene) and KMT1 gene, which is used for confirmation of Past. Multocida identification with PCR (Townsend et al., 2001). Also, Ranjan et al. (2011) described that toxA gene could be useful for direct analysis of toxigenic Past. multocida. In this study, the characteristic bands for both genes were observed at 460 and 864 bp in all tested isolates. El-Shehedi et al. (2016) reported Tox A gene association with the diseased status of the animal.

The obtained results of antimicrobial sensitivity tests revealed that the high resistance rates of E. coli isolates against amoxicillin clavulanic acid, penicillin, ciprofloxacin and sulphamethoxazole (100% each).  In the same way, 100% resistance rate of E. coli isolates against sulfamethoxazole and amoxicillin clavulanic acid was recorded with (Ammar et al., 2015). Penicillin and norofloxacin exhibited high resistance levels (100% of each) against the tested S. aureus isolates and (90%) for tetracycline Similarly, Abd-Al-Azeem et al. (2013) stated that the highest resistance of β-lactam resistant S. aureus isolates was for penicillin and the lowest was for amoxicillin clavulanic acid. All tested strains of Past. Multocida were highly sensitive against the mostused antibiotics except low resistance was recorded against tetracycline (41%) followed by enerofloxacin and norfloxacin antibiotics (30%). Similarly, Carty et al. (2005) reported that gentamicin was highly effective (87.5%) against Past. multocida isolates and they recorded their acquired resistance to enrofloxacin. El-Shehedi et al. (2016) indicated the high sensitivity of Past. Multocida isolates to ciprofloxacin, gentamicin and moderate sensitivity to enerofloxacin and norfloxacin.

The phenotypic resistant pattern was in parallel to the genotypic detection of their antibiotic resistance genes. The phenotypic resistance of E. coli isolates to amoxicillin-clavulanic acid and ciprofloxacin compounds could be explained by the presence of bla_TEM and qnrS resistance genes among the twenty examined isolates. Colom et al. (2003) and Eid and Erfan, (2013) recorded the high incidence rates of bla_TEM gene in E. coli isolates were previously recorded in Spain and Egypt 88% and 79%, respectively. Tetracycline resistance is generally caused by the acquisition of a tetracycline resistance (tet) gene, as these genes are associated with primary resistance mechanisms, which involve active efflux pumps, ribosomal protection, and enzyme inactivation (Koo and Woo 2011). In the present study, tetA was found in 80% of E. coli isolates. This result was in paralleling with Sengelov et al. (2003) who elucitad thattet (A) gene was the most abundant (71%) of 100 E. coli isolates from diseased and healthy pigs, cattle and broiler chickens.

The multidrug efflux pump NorA is one of the most studied efflux systems in S. aureus. Increased resistance to fluoroquinolones had been associated with NorA-mediated efflux, via the increased expression of the norA gene (Costa et al., 2013). NorA, in this study, is exhibited in 100% of S. aueus isolates that were phenotypically resistant to norofloxacin. However, blaZ gene (encodes for β-lactamase); have been frequently reported in many isolates of S. aureus (90%). It acts through hydrolysis of the peptide bond in the β-lactam ring (Jensen and Lyon, 2009). Yang et al. (2005) and Martini et al. (2017) recorded that 94.6% and 97% carried blaZ gene respectively among the penicillin resistant S. aureus isolates.

The observed respiratory signs in our study come in parallel with Radostitis et al. (2007) and Smith, (2015), which attributed to excessive formation and accumulation of ammonia especially in bad ventilated houses, leading to irritation and inflammation of mucous membranes inducing nasal discharge, and dramatic abnormal rales.

The effect of pneumonia on red cell parameters were significant decrease in RBCs count, Hb, PCV, MCV, MCH and MCHC values in the diseased group indicating the presence of microcytic hypochromic anemia. The hematological examination showed anemia in pneumonic bovine calves that might be attributed to the destruction of red blood cells by microorganism secretions (Mondal et al., 2004). Anemia may be due to anorexia observed with pneumonia or sequestration of iron in bone marrow macrophages and hepatocytes during infection, thus become unavailable for utilization in hemoglobin synthesis leading to inhibition of erythropoiesis (Mosa, 2000). Decreases iron transfer into developing erythroid cells in bone marrow leading to reduction of Hbsynthesis and production of microcytic hypochromic RBCs (El-Naser and Khamis, 2009 and Aytekin et al., 2011).

Moreover, the significant increase of TLC and neutrophils might be attributed to inflammatory lesions and presence of bacterial infection (Abou El-Gheit, 2000). On the other hand, the significant decrease of lymphocytes might be attributed to the stimulation of adrenal gland during stress with the tissue invaded by bacterial toxins (Abou El-Gheit, 2000). Eosinophilia could be the result of E. coli and Staph.aureus microorganisms (Raghib et al., 2004).

The significant increase of serum enzymes AST and ALT levels in our study might be attributed to the degenerative and necrotic changes in liver and kidney accompanied the formation of pulmonary lesion due to bacterial infection and its toxin. These agree with Abou ElGheit (2000) and Saleh and Allam (2014). Higher CK activity was recorded in infected calves probably as a result of increased breathing rate and increased muscle activity in the course of prolonged duration of severe respiratory disease or consequence of dystrophic damage of muscles during longer lasting recumbency. Elevation of the AST and CK activity was also observed by Abdullah et al. (2013).

The significantly (P<0.05) increased in urea concentration could be explained by the accelerated catabolism of body protein to compensate anorexia and could be due to infection, while the significant (P<0.05) increased in serum creatinine might be attributed to kidney dysfunction after infection (Radostits et al., 2000).

The observed highly significant decreased (P <0.01) in total proteins might be attributed to isolated bacteria or bacterial toxins that increase capillary permeability escaping of plasma proteins into tissues, (Omran et al., 2005). However, the observed highly significant (P <0.01) decreased albumin level was agreed with Civelek et al. (2007) and El-Deeb, (2011). Furthermore, it could be attributed to anorexia associated with pneumonia and inability of liver to synthesize protein. The hyperglobulinemia might be due to the stimulation of immune system by the infectious agent (Abd El-Raof and Hassan, 1999). The A/G ratio in the pneumonic calves was significantly lower. In our study, comparison of serum protein fractions between healthy and diseased animals showed significantly higher concentrations of α1-globulins in the pneumonic calves. Cerón et al. (2011) indicated that increased α-globulin concentrations were indicative of acute inflammation. Tymchak, (2010) reported that chronic infections might produce an increase not only in globulin fractions, predominantly in γ-globulins, but also in α- and β-globulin fractions, which was accompanied by a decrease of albumin. In addition Stockholm and Scott (2008) elucidated that decreased albumin and increased globulin concentrations were the most common pattern in animals with inflammatory diseases. This shift in albumin and globulin concentrations resulted in significantly lower A/G ratio in calves affected by chronic respiratory diseases. In our study in diseased calves had significantly lower mean concentration of serum glucose. This may be due to longer lasting inadequate feed intake and energy supply during times of illness. Decreased concentrations of glucose were reported by Hanzlicek et al. (2010) during experimentally induced pneumonia in calves.

The analytic study indicated significantly lower mean values in the serum concentrations of Mg, P, and Fe in diseased calves. Fraser et al. (2014) found a significant decrease of K and P concentrations in Mycoplasma bovis and in Ca and Na concentrations in calves after experimentally induced pneumonia. Significant differences between healthy calves and calves with pneumonia with lower values of Ca and K in diseased animals were recorded by Ragbetli et al. (2010). Hanzlicek et al. (2010) found evident changes in the concentrations of K (decrease of values) in pneumonia. The decrease in serum calcium might be the result of anorexia, decreased intestinal absorption or increased renal excretion (Radostitis et al., 2007). Approx. 40-45% of calcium is protein bound mainly to albumin, so hypoalbuminemia might be a possible cause for this hypocalcemia (Kaneko et al., 2008). The significant decrease in serum phosphorous concentrations seemed to be secondary to reduced phosphorus absorption from the gut and reduced phosphorus resorption from the tissues (Orr et al., 1990).The significant decrease in serum iron (Fe) in pneumonic calves agrees with Blum et al. (1996) who revealed decreased concentrations of blood plasma iron in calves with chronic pneumonia. These decreased Fe could be due to reduction of energy and protein intake or sequestration of iron in bone marrow macrophages and hepatocytes during infection, (Kaneko et al., 2008). Decreased appetite might explain the lower serum concentrations of phosphorus and magnesium in calves, both are much more dependent on dietary intake (Rowlands, 1980).

Fibrinogen is considered a consistent marker of bacterial infection and inflammation in domestic ruminants (Youssef et al., 2015). Significantly higher values of plasma fibrinogen (F) concentrations were detected in the diseased calves compared to the respective control group. This elevation may be attributed to the involvement of F in modulating hemostasis, inflammatory response, and the tissue repairing process (Feldman et al., 2000).

In conclusion, this study provided a nucleus of information regarding bacteria encountered in the upper respiratory tract of calves. Although they are normal flora in the upper reparatory tract, they may help in the progress of calves’ pneumonia especially if they are accompanied with presence of risk factors. Alteration hemato-biochemical parameters could be useful diagnostic tools treatment for pneumonia in cattle calves. The study of a correlation between the phenotypic and genotypic antimicrobial resistance and virulence genes among the recovered isolates could be effective for understanding the dangerous spread of virulence genotypes and antibiotic resistance of these species. Recommendations for minimizing the non-responsible use of antibiotics for treatment of pneumonia in calves’ farms should be applied to avoid more dissemination of the multidrug-resistant bacteria.

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