Abstract: Background: Pneumonic pasteurellosis is an emerging disease cause gigantic losses among calves. Objective: The present study was designed to study the different methods for diagnosis of the disease with special reference to the role of vitamin D in occurrence of the disease. Materials and Methods: Ninety seven buffalo calves were used in this study, 10 calves were clinically healthy (control group), other 87 calves were diseased. All calves were subjected to thorough clinical and ultrasonographic examination. In addition two blood samples were taken for hematological studies and all sera were tested by commercial kits for biochemical analysis. Bacteriological studies and detection of virulence-associated genes by PCR were applied. Results: Hematobiochemical revealed leucocytosis with neutrophilia. A significant decrease in serum glucose, total proteins, albumin and vitamin D. Pulmonary lesions were diagnosed ultrasonographically. Pulmonary parenchyma appeared homogenous hypoechoic structure "Hepatinization" with pleurisy as there is an accumulation of anechoic fluid between the parietal and visceral pleura. Nineteen isolates of Pasteurella multocida were isolated from nasal swabs and pulmonary tissues with antigenic typing belonging to type B2 and the pathogenicity test revealed that 100% mortality of injected mice with different time intervals ranged from 24-72 h. The high prevalence of Pasteurella filamentous haemagglutinin A (PfhA) and transferrin binding protein encoding (tbpA) genes with prevalence rate (46.7 and 94.9%), respectively, among the isolates point toward the positive association of these virulence genes with the occurrence of the disease in buffalo calves. Postmortem examination of the dead animals showed pulmonary congestion, edema, consolidation and hepatization of some pulmonary lobes. Fibrino broncopneumonia and edema with infiltration of inflammatory cells are the most prominent finding histopathologically. Conclusion: This study suggests that decrease of vitamin D has a role in occurence of the diseases. In addition Pasteurella multocida is highly susceptible to enrofloxacin, sulphamethoxazole and nitrofurantoin than other antimicrobials.
INTRODUCTION
Bovine Respiratory Disease Complex (BRDC) is common in calves and yearlings, causing gigantic economic losses due to reduced live weight gain and feed conversion efficiency, poor performance and cost of treatment in addition, it is considered the most common reason for calf deaths in Egypt and around the world. One of main causes of BRDC is pasteurellosis1,2. It is an important infectious disease of ruminants causing great economic loss with high morbidity and mortality. Pneumonic pasteurellosis is triggered by stress factors as poor weather and management, humid conditions, transportation, mixed age groups, high stocking densities and previous respiratory infection3. As these stress factors reduce the immune system allowing Pasteurella species which are commensally inhabitant in the respiratory tract of healthy ruminants as normal constituents of the nasopharyngeal microflora to invade the respiratory tract causing infection and damage to lung tissue in animals with a compromised pulmonary defense system.
Clinically, the disease characterized by short course. The diseased calves usually exhibit clinical signs of acute respiratory distress. Although, in some cases sudden death appears before the development of significant pulmonary lesions. The infected animals appear extremely dull, depressed, lethargy with reduced appetite. They soon develop a high fever, anorexia and abnormal breathing pattern (rapid shallow respiration) accompanied with profuse mucopurulent nasal and ocular discharges. Later on, productive cough. Marked dyspnoea with an expiratory grunt may present. Auscultation of the lung fields bilaterally may reveal crackles and wheezes4.
The both circulating form of vitamin D, 25-hydroxyvitamin D (25(OH) D) and its active form, 1,25-dihydroxyvitamin D (1,25(OH)2D) were originally known as important endocrine hormones in calcium homeostasis and bone health. However, recent studies during the last two decades suggest the main role of vitamin D in endothelial function, hematopoietic, cell differentiation, proliferation and immune function. It appears to be an integral component of the immune system as it is essential for innate and adaptive immune system including macrophages and lymphocytes5,6. It plays a role in gene expression in immune cell and increases their ability to kill pathogens. As vitamin D receptor responds to 1,25(OH)2D and regulates antimicrobial peptides cathelicidin and beta-defensing7. Based on these data vitamin D insufficiency is associated with increased incidence of respiratory tract infections8. Levels of vitamin D tend to decrease in the winter. So, lower level of vitamin D may be a factor in why that infectious diseases such as flu and pneumonia tend to be more common in the winter9.
Ewers et al.10 showed that Pasteurella filamentous haemagglutinin A (PfhA) and transferrin binding protein encoding (tbpA) genes act as important epidemiological marker genes for characterizing P. multocida field strains in addition to the association with bovine diseases. The tbpA protein is necessary for extraction of iron from transferrin11.
History and clinical signs aid in the diagnosis of pneumonic pasteurellosis, however, further investigation are required to confirm the diagnosis. So, this study was aimed to throw light on the clinical signs, hematology, bacterial isolation, pathologic and ultrasonographic alterations associated with pneumonic pasteurellosis thus to assist veterinarians in recognizing cases in the field and to define the better extent of disease. With special reference to correlate the relationship between the immunological role of vitamin D and pneumonic pasteurellosis.
MATERIALS AND METHODS
Animals: A syndrome of calf pneumonia of feedlot calf herds was observed in 2 farms in Ismailia province "El-Salheia city". The ages of calves ranged from 7-12 months. This study was performed on 87 buffalo calves, 10 calves were clinically healthy of the same age and reared under the same conditions of the diseased ones considered as a control group in addition to 6 tissue samples were collected from emergency slaughtered cases.
Clinical examination: A complete case history and farm complaint were recorded. All animals were subjected to thorough clinical examination including taking the rectal temperature, respiration, auscultation of the heart and lung with examining the mucous membrane and superficial lymph nodes. Calf respiratory scoring was conducted according to the Wisconsin Calf Scoring Chart (WCSC), this system was described by McGuirk12.
Blood sampling: Two blood samples were taken from each animal via puncture of the jugular vein. The first blood sample was collected in vacutainer tubes containing EDTA. An automated cell counter was used to measure total and differential leukocyte count. The second blood sample was collected in plain vacutainer tube for separation of serum13. The harvested sera were transferred into eppendorf tubes, which were coded and kept at -20°C until the time of analysis. Serum vitamin D level was measured using Elisa kits (ABIN858623 inf@antibodies-online.com). The serum glucose, total protein, albumin and globulin were estimated by standard procedures using (Diagnostic Zrt., Commercial kits) which were provided by Egyptian company for biotechnology, Cairo, Egypt and the reading was taken by the spectrophotometer.
Ultrasonographic examination: The same area that was auscultated was systematically scanned ultrasonographically focused on the 4th-8th intercostal space. Both sides of the thorax were screened for the presence of abnormal ultrasonographic findings14,15. Ultrasonography was performed using a 5 MHz probe that was directly applied on the thorax after application of coupling gel to improve image quality.
Bacteriological studies: Samples for bacteriological examinations include 97 nasal swabs, 87 were collected from diseased calves and 10 from apparently healthy ones in addition to 6 tissue samples were collected from emergency slaughtered cases were taken from lung and trachea for aerobic examinations. All samples were collected in sterile polyethylene bags as well as nutrient broth as transport media for further bacteriological examination.
Bacteriological examination: Under aseptic conditions from the entire of the collected samples were inoculated into a nutrient broth and then cultured on nutrient agar, blood agar and MacConkey agar plates. The inoculated plates were incubated at 37°C for 24 h. Suspected colonies were characterized on the basis of colony morphology and pure colonies were identified biochemically according to Koneman et al.16.
Pathogenicity and virulence of isolated P. multocida: Nineteen P. multocida isolates were tested for the pathogenicity and virulence as a more common isolate, 5 Swiss Webster white mice weighting about 18-22 g were used for each isolate. All mice (95 mice) were injected intraperitoneally (i.p.) by 0.1 mL of the bacterial suspension of 1.5×108 CFU. One mouse was kept as a control for each isolate and was injected i.p., with 0.1 mL sterile normal saline. The mortality rates and post-mortem changes were recorded. From heart blood of dead mice, reisolation of inoculated strains was carried out and blood films were prepared and stained with Gram stain for showing the characteristic features of P. multocida organisms17.
Serotyping of P. multocida isolates: Isolates were analyzed by using rapid slide agglutination test using capsular type B antiserum according to Rimler and Rhoades18. Serotyping was performed in the Department of Clinical Microbiology, Central Health Laboratories, Ministry of Health, Cairo, Egypt.
Detection of virulence-associated genes by PCR: The sequences of oligonucleotide primers, amplification conditions were showed in Table 1 and the PCR reactions were adapted according to Ewers et al.10 in which 0.4 μL of bacterial culture stock, without genomic DNA extraction step were taken as DNA template and added to the reaction mixture (25 μL) containing 2.5 μL 10X PCR buffer, 2.5 μL of 25 mM magnesium chloride, 0.4 μL of 25 mM dNTPs, 0.5 μL of each primer pair in a 10 pmol concentration and 1 U of Taq-polymerase (Promega corporation, Madison, USA). The samples were then subjected to 25 cycles of amplification in a thermal cycler (Applied Biosystems, USA). Amplification of products was analyzed by gel electrophoresis on a 1% agarose gel, stained with ethidium bromide and photographed at UV exposure.
Antimicrobial susceptibility test: The susceptibility profile of P. multocida was tested according to the procedures of NCCLS19 using disk diffusion technique. Pure colonies from the blood agar medium, incubated at 37°C for 18 h, suspended in 2 mL sterile saline to a density approximately equal to Mc-Farland opacity standard No. 0.5. A dry cotton wool swab was placed into the suspension and excess liquid was expressed against the inside of the tube. The bacterial suspension was then inoculated on to Mueller-Hinton agar with the swab in such a way that the whole surface of the agar was covered.
| Table 1: | PCR primers and PCR conditions used for the detection of two virulence associated genes |
The plates were recorded by measuring the inhibition zone diameter according to the interpretive standards of NCCLS. The following 9 antimicrobial agents were assayed: Fusidic acid (10 μg), amikacin (10 μg), enrofloxacin (5 μg), gentamycin (10 μg), cefoxitin (10 μg), kanamycin (10 μg), tetracycline (30 μg), trimethoprim/sulphamethoxazole (25 μg) and nitrofurantoin (10 μg).
Necropsy procedure and histopathological examination: The emergency slaughtered cases were necropsied and gross lesions were recorded. Tissues were fixed in 10% phosphate-buffered formalin then sections were stained with hematoxylin and eosin and examined microscopically.
Statistical analysis: Data were analyzed using packaged SPSS program. All data were presented Mean±Standard Error (SE). Differences between groups were determined by LSD post hoc test. The significance level was set at p<0.05.
RESULTS
Epidemiological data: A syndrome of calf pneumonia of feedlot calf herds was observed in 2 farms in Ismailia province "El-Salheia city". The ages of calves ranged from 7-12 months. This study was performed on 87 buffalo calves, 10 calves were clinically healthy considered as a control group in addition to 6 tissue samples were collected from emergency slaughtered cases. The cases started to appear in the herds about 1 weeks after animal transport. Within a month, about 87 calves out of 190 showed clinical signs of pneumonia (morbidity was 46%) and the mortality was 3.2% (Table 2).
Clinical signs: Calves showed signs of dry, hacking cough, depression, fever accompanied with profuse mucopurulent nasal and ocular discharges, labored breathing and mouth breathing. Some cases there was profuse salivation. Later on, productive cough as illustrated in Table 3.
| Table 2: | Incidence of pneumonia in examined buffalo calves |
| Table 3: | Physical examination including numerical respiratory score of apparently healthy and diseased calves according to the Wisconsin Calf Scoring Chart (WCSC) |
| Fig. 1(a-e): | (a) Normal ultrasonographic image of lung and pleura. Echogenic costal and pulmonary pleura with reverbration artifact medial and parallel to pleura with diffent echogenicity, (b-c) Pneumonic lung, replacement of reverbration artifact by hypoechoic structure similar to that of liver "Hepatiztion", (d) Pleuroneumonia revealed by thickening of pleura and (e) Prescence of anechoic fluid in between the pleural layers with change of pulmonary parenchyma into homogenous hpoechoic structure "5 MHz" |
| Table 4: | Statistical-evaluation of some hematobiochemical parameters in clinically healthy and pneumonic calves |
| NS: Non-significance | |
All diseased calves suffered from reduced appetite to anorexia, rapid shallow respiration (tachypnea "32.1±0.19" with tachycardia "95.8±4.31"). Auscultation of the lung revealed exaggerated vesicular sound, dry rales and in some cases heart sound heard on the lung. The course of the disease is short lasting a few hours to a few days and most animals die with septicemia in the terminal stages. Recovery rate from the clinical disease is rare and the curing occurs only when animals are treated early in the initial stages of the disease.
Haematobiochemical analysis: Revealed leucocytosis with neutrophilia. Hypoproteinemia and hypoalbuminemia with hyperglobulinemia (Table 4). A significant decrease of vitamin D in diseased calves compared with the control ones.
Ultrasonographic examination: Lung of diseased calves ultrasonographically revealed different abnormalities in the form of presence of hyperechoic inflammatory cells in the parenchyma of the lung "Pneumonia". Lung consolidation appeared as a homogenous hypoechoic structure similar to the hepatic cell as in (Fig. 1b, c). Another abnormality was pleural effusion as pleural fluid accumulation was diagnosed between the parietal and visceral pleura. The pleural irregularity was noted, in contrast to a smooth hyperechoic line as in (Fig. 1d, e), the pleural line was serrated with an irregular shape.
Isolation and serotyping of Pasteurella multocida: Isolation and identification of P. multocida in the present study revealed that Gram-negative, bipolar-staining short bacilli. Pasteurella multocida organisms produce catalase, oxidase, indol and reduce nitrates.
| Fig. 2: | Blood film showing the bipolar organisms of Pasteurella multocida "Arrow" when stained with Gram stain |
| Table 5: | Virulence and pathogenicity of isolated Pasteurella multocida in mice |
They do not produce urease or hydrogen sulfide and fail to liquefy gelatin or use citrate. Sucrose and glucose are always fermented with the production of acid only.
Serotyping: Identification of P. multocida isolates by using rapid slide agglutination test using capsular type B antiserum indicate P. multocida type B2.
Pathogenicity of P. multocida: Results in Table 5 revealed that isolates were tested experimentally in white mice by injection of 0.1 mL of P. multocida broth culture in 5 mice for each isolate. The death of the inoculated mice usually occurs within 24 h. The number of tested isolates was 19 isolates from P. multocida. All dead mice showed generalized septicemic appearance with highly congested subcutaneous tissues and internal organs, reisolation of inoculated organisms from heart blood of dead mice was carried out. The prepared blood films showed bipolar organisms of P. multocida when stained with gram stain (Fig. 2).
Virulence genes detection using PCR analyses: Virulence genes was detected by amplification of two virulence factor genes using multiplex PCR in the presence of specific primers. A total of 19 P. multocida isolates belonging to capsular type B harbored the virulence associated genes: tbpA and PfhA with prevalence rate (94.9 and 46.7%), respectively. Amplification of DNA bands with about 275 and 728 bp sizes have illustrated the presence of PfhA and tbpA genes in these isolates, respectively.
Antibiotic susceptibility test: In Table 6 all tested isolates were highly sensitive to enrofloxacin, sulphamethoxazole and nitrofurantoin (100%). With variable sensitivity to fusidic acid (84%), gentamycin (74%), kanamycin (63%) and tetracycline (37%). On the other hand, all isolates were highly resistance to amikacin and cefoxitin (100%).
Gross pathology: Post mortem examination of slaughtered calves revealed that the lungs of dead calves showed fibrinous bronchopneumonia with prominent fibrinous pleurisy and pleural effusions. Subcutaneous, interstitial and widespread petechial hemorrhages with congested lungs (Fig. 3a). The diseased portions of the lung become remarkably consolidated or hepatized (Fig. 3b, c) with a marked thickening of the interlobular septa due to fibrinous edema giving marbling appearance (Fig. 3d).
Histopathological examination: Lung tissues revealed marked pulmonary edema in some lobules and fibrinous exudate in other alveoli (Fig. 4a) and its infiltration with leukocytes particularly polymorph nuclear cells and few monocytes.
| Fig. 3(a-d): | (a) Lung of buffalo calf showing congestion, (b) Edema, (c) Consalidation "Arrow" and (d) Marbling appearance |
| Fig. 4(a-b): | Histological appearance of the lung of a buffalo calf that died of haemorrhagic septicaemia. (a) Marked pulmonary edema with partial obstruction of the lumen of a medium sized bronchus with fibrin plug (arrowheads) (H and E 50X) and (b) Marked obliterations of the alveolar lumina with fibrin threads (arrowheads) and infiltrated with leukocytes (H and E 100X) |
Partial to complete obstruction of the lumina of the medium sized bronchi with fibrin plugs that are usually infiltrated with leukocytes were seen (Fig. 4b).
| Table 6: | Antibiotic sensitivity of 19 isolates of Pasteurella multocida |
DISCUSSION
Pneumonic pasteurellosis is a disease that mainly occurs in animals with a compromised pulmonary defense mechanism. The incidence of the disease was high in calves more than 3 months. Stress and/or viral infection impair the local pulmonary defense mechanisms causing bad effects on the ciliated cells and mucous coating of the respiratory tract causing suppression of the mucociliary clearance mechanism. Pasteurella multocida is commonly present in the upper respiratory tracts as an opportunistic or commensally normal inhabitant pathogen20. It was isolated from lung tissues of the slaughtered cases. This may refer to the presence of a risk factor induces a favorable media in lung facilitating the invasion of P. multocida into deep tissues. Despite P. multocida being nasopharyngeal microflora21, when invading lung tissue under stress factors, its virulence exaggerates and pathogenicity differs22. Endotoxins produced by rapid growth and multiplication of Pasteurella in the infected lobules will cause extensive intravascular thrombosis of pulmonary veins, capillaries and lymphatics. These vascular disturbances result in focal ischaemic necrosis of the pulmonary parenchyma accompanied by severe inflammatory reaction dominated by fibrinous exudate 23.
The incidence of p. multocida isolated from nasal swabs (20.7%) while in lung about 83%. The results revealed that P. multocida was isolated from pneumonic calves with a percentage (20.7%). These results agree with Fulton et al.24, Ranjan et al.25 and Enany et al.26. In comparison with previous studies, low incidence from buffalo calves with pneumonic pasteurellosis27 15.89 and 8% by Novert1. The variation in isolation percentage may be due to the difference in the hygienic measure used, stress factors, change in management and immunity of infected animals28.
Our clinical signs were previously described by Abubakar and Zamri-Saad29 and Abdullah et al.30. Auscultation of the lung revealed exaggerated vesicular sound, dry rales and in some cases heart sound heard on the lung, the results of this study agreed with that recorded by Abdullah et al.30 and Mohamed and Abd El-Salam31. Marked dyspnoea with an expiratory grunt observed in very advanced stages of the disease23. Fever is an indicator of microbial infection. It is one of the mechanisms that immune system utilizes to get rid the body from invading pathogens32 and as a normal response to an immune stimulus. The change in attitude, appetite, rectal temperature and mucous membrane congestion of the animal may be attributed to toxemia which associated with the release of the bacterial endotoxin.
Leucocytosis with neutrophilia may be attributed to the bacterial infection. Maunsell et al.33 recorded neutrophilia with the subsequent release of large amounts of inflammatory mediators in bovine respiratory diseases. There is a relationship between the neutrophil production and lung injury causing the release of some enzymes such as elastase and myeloperoxidase (MPO) and Nitric Oxide (NO) generation which considered a strong weapon against the invading microorganisms as neutrophil is an essential first-line responder to the bacterial infection acting as a clearance of the bacteria from the affected tissue34. In bacterial infection, neutrophilic infiltrates in the bronchial, bronchiolar and alveolar compartments of the lung are the main pathological change associated with bronchopneumonia. The recorded hypoproteinemia, hypoalbuminemia with hyperglobulinemia were attributed to anorexia of the diseased calves, the inability of their liver to synthesize the protein and also may be due to the bacteria and its toxins which increase the permeability of the blood capillaries causing the escape of the protein.
Vitamin D plays a role in the regulation of immunity, both innate and adaptive immunity35 as vitamin D receptors (VDRs) present in most cells of the immune system. Vitamin D has another defensive role in regulating the inflammatory response and chemo-attracting cells of the adaptive immune system to sites of infection. It binds and neutralizes the lipopolysaccharides produced from the bacterial wall and enhance the re-epithelization.
The lower level of vitamin D in diseased calves compared with the control ones, present results were agreed with Ginde et al.36 who stated that lower serum vitamin D level is associated with increased risk of upper respiratory tract infection and associated complications. As when toll-like receptors on macrophages bind the bacterial wall lipopolysaccharides (LPS), leading to the increase of 1-α-hydroxylase and vitamin D receptor expression is increased37 resulting in local conversion of inactive vitamin D to the active form, which in turn increases cathelicidin and beta-defensing "Antimicrobial peptides" and bactericidal proteins which act as natural antibiotics combating the infection. Also, Cannell et al.38 showed that vitamin D deficiency is associated with pneumonia in calves, so supplementation of this vitamin would give the animal more resistance against the diseased associated with stress factors affecting the immune system.
Ultrasonographic examination of the lung revealed different abnormalities in the form of presence of hyperechoic inflammatory cells in the parenchyma of the lung "Pneumonia". As neutrophilic infiltrates in the bronchial, bronchiolar and alveolar compartments of the lung are the main pathological change associated with bronchopneumonia39 which effectively displace air from the lung tissue, resulting in the lung consolidation giving hypoechogenic structure similar to that of liver "Hepatinization". The results of this study were similar to that of Buczinski et al.4. Although, the previous experimental studies using P. multocida-induced pneumonia have shown that the clinical signs, ultrasonographic lesions and spirometric signs are not closely correlated14.
The virulence and pathogenicity test of P. multocida in mice revealed that all isolates were highly pathogenic to mice producing acute septicemia and death within 24-48 h post inoculation. Small doses of P. multocida recovered from nasal swabs were highly virulent and sufficient to kill all mice 40,41. In the present study, the failure for bacteriological isolation in one lung and nasal swab samples may be due to other cause incriminated as viruses, fungi or mycoplasma. As the etiology of pneumonia is complex and multifactorial which are either microbial determinants including bacteria, viruses, fungi or non-infectious.
Veken et al.42 reported that the prevalence of PfhA and tbpA genes among P. multocida type B in bovine isolates associated with pneumonia. The prevalence of 2 virulence-associated genes which included the gene coding for iron acquisition factors (tbpA) and adhesion related gene (PfhA) were carried out in which our isolates possessed tbpA and PfhA with prevalence rate (94.9 and 46.7%), respectively. In contrary to Verma et al.11 who reported that which all isolates had tbpA and PfhA genes in both diseased and healthy one, however their presence in isolates that isolated from healthy bovines need more investigation about their role in disease production. On the other hand, the result was in agreement with Ewers et al.10 and Sarangi et al.43 who reported the high prevalence of tbpA gene and the low prevalence of PfhA with frequencies ranged between 46-52%. In present result in spite of 100% mouse lethality test of 19 isolates, only 94.9% possessed tbpA and 46.7% had PfhA genes which may be attributed to the difference in gene expression between in vivo and in vitro or the presence of defective and non function genes which can not be detected.
Pasteurella multocida isolates were highly susceptible to enrofloxacin, sulphamethoxazole and nitrofurantoin (100%). On the other hand, all isolates were highly resistance to amikacin and cefoxitin (100%). This result was in contrary with Elshemey and Abd-Elrahman44 who showed that P. muItocida isolates were sensitive to gentamycin (84%), while less sensitive to enrofloxacin (72%) but P. multocida isolates were resistant to tetracycline and trimethoprim-sulfametoxazol. In addition variability of sensitivity also detected by Wassenaar and Silley45 as most P. multocida isolates were resistance to tetracycline, oxacillin, streptomycin and trimethoprim. Also 67% of isolates were resistant to cephalexin46. The majority of P. multocida isolates were sensitive to enrofloxacin, gentamicin and choramphenicol but moderately sensitive to ciprofloxacin and pefloxacin and were resistant to oxytetracycline, streptomycin, amoxycillin, tetracycline and sulphadimidine11. We thought that the variability of our isolates to antibiotic sensitivity was regard to huge use of the different antimicrobial in addition to locality were the isolates detected.
A similar result of pulmonary lesions and histopathological examination were previously reported by Abdullah et al.30, Mohamed abd Abd El-Salam31 and Odugbo et al.47. The severely pulmonary lesions may be attributed to lysis of bovine alveolar macrophages and neutrophils within the alveoli which induced by bacterial leukotoxin.
CONCLUSION
It is obvious from the present study and previous literature that pneumonic pasteurellosis is a highly complex multifactorial disease of a worldwide prevalence. Calves are more affected, the disease primarily results from the interaction of stress, decrease the immunity and the causative bacteria "Pasteurella multocida type B2" causing pneumonia in buffalo calves in age ranged from 7-12 months. The high prevalence of Pasteurella filamentous haemagglutinin A (PfhA) and transferrin binding protein encoding (tbpA) genes may be associated with other predisposing factor in production of the disease in buffalo calves. In addition, it is implicated that vitamin D has an important role in raising the immunity of the animals and decrease the susceptibility to respiratory affection. Antimicrobial drugs either enrofloxacin or sulphamethoxazole or nitrofurantoin should be included in the treatment of active cases.
ACKNOWLEDGMENT
Acknowledgment is gratefully made to the owners farms for their assistance and cooperation during the collection of samples.