bacteriological, mycoplasmal, virological and pathological … (77-122).pdf · 2011-05-10 ·...

Egypt. J. Comp. Path. & Clinic. Path. Vol. 21 No. 2 (April) 2008; 77– 122

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Bacteriological, Mycoplasmal, Virological and Pathological Studies on Mortalities in Imported Cattle

By *Fatma, M. Darwish; **Soumaya, S.A. El-Shafii, ***Omayma A.S-

hemies and ****Zeinab, R. Mohamed *Pathology Department, **Bacteriology Department; ***Virology

Department and ****Mycoplasma Department. Animal Health Research

SUMMARY

M ortalities in two imported Frisian cattle herds were investigated within two months after arrival in governmental quarantine at Suez governorate in 2006

and 2007. The mortality rate was 8.34% in herd (1) 9.02% in herd (2) respectively. The

diseased animals in the two herds showed respiratory manifestations, lacrimations, ptyalism, mucopurulent nasal discharges, depression recumbency, anorexia and pro-fuse watery diarrhoea.

The dead calves were necropized, gross lesions were reported and tissue specimens from internal organs were selected as well as nasal, ocular and rectal swabs were obtained from the diseased animals for histopathological, virological, bacteriological and mycoplasmal studies. Moreover, serum samples and blood sam-ples on anticoagulant were collected from diseased and apparently healthy animals for virological and serological identification.

On virological studies, AGPT and commercial ELISA kits were rapid and ac-curate tests for detection of BVDV antigen. BVDV was isolated on MDBK cell line from buffy coats, nasal swabs and rectal swabs collected from diseased calves and from internal organs of dead ones, and was identified by IFAT using reference antis-era. Also 64 serum samples collected from apparently healthy and diseased calves were tested by VNT for the detection of neutralizing antibodies against BVDV.

Moreover, the lungs, livers, kidneys of dead calves as well as nasal swabs of diseased ones yielded Mannhemia haemolytica on bacteriological investigation. The isolated strains were biotyped to biotype A ( 69 isolates) and biotype B (6 isolates). On virulence test, most M. haemolytica isolates were highly pathogenic for mice, where mortality occurs within 12-48 h. Meanwhile, the resistance of the isolates to most antimicrobial agents was high (resistant to ceftiofur, nalidixic acid, oxytetera-cycline, cephaloridine, gentamicin, cephalexin and donax and highly sensitive to norfloxacin, ampicillin and erythromycin).

Meanwhile Mycoplasma bovis were recovered from trachea, lungs, and livers of dead calves and nasal swabs of diseased ones.


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Histopathological studies of dead calves depicated fibrinointerstitial bronchopneumonia, hepatocellular necrosis, necrobiotic changes of cardiac myocytes, immunecomplex glomerulonephritis (proliferative and membranous forms), lymphofollicular depletion of lymphoid cells in the spleen and mesen-teric lymph nodes, necrotic changes of the ruminal and reticular mucosa, ne-crotizing enteritis with focal erosions and /or ulceration of the mucosa. The pre-sent investigation explored out that the principle cause of deaths was BVD viral infection which predispose and act synergistically with M. haemolytica and M. bovis through its immunosuppressive effect.

Referred byReferred by Prof. Dr. Ahmed M. Ammar Professor of Microbiology, Fac. Vet. Med.,

Zagazig University Prof. Dr. Afaf El-khwas Professor of Pathology, Animal Health Re-

search Institute, Dokki Prof. Dr. Nawal M. Ali Professor of Virology, Animal Health Research

Institute, Dokki

INTRODUCTION

M ortality in imported cattle is of great impact on beef indus-

try since it has direct adverse effects on production (Chi et al., 2002).

Most deaths in imported cattle occurred shortly after arrival and mainly in attribute to respiratory (shipping fever pneumonia, diges-tive or other disorders which are a consequence of interactions among stressors, host immunity, patho-genicity of infectious agents and en-vironmental factors that alter the probability of pathogen exposure (Loneragan et al., 2001 and She-hab et al., 2001).

Beef industry suffers large economic losses from various infec-tious agents which hinder animal productivity represented by prema-ture culling of infected animals, re-

duced slaughter value of the meat and reproductive losses (Chi et al., 2002 and Hägglund et al., 2007).

One of the most important in-fectious agents which infects cattle shortly after entering to the feedlot is BVD virus (Brodersen and Kel-ling, 1998). BVD virus has been as-sociated with pathology of several physiologic systems including diges-tive, hematologic, immunologic, neurologic and reproductive systems (Grooms, 2004). Moreover, BVD virus is accorded a role in the causa-tion of respiratory tract diseases and cause immune suppression as it acts synergistically with other pathogens results in more severe diseases.

BVD is a positive stranded RNA virus of the genus Pestivirus within family Flaviviridae (Ra-mirez et al., 2001). Two biotypes,


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non-cytopathic (ncp) and cytopathic (cp) are differentiated by their effect on suscept ib le ce l l cul tures (Beknopet, et al., 2000).

The characteristic features of the Pestiviruses are the ability of the placental transmission of ncp-BVDV to infect the fetus at a par-ticular stage of gestation resulting in persistently infected (PI) calves (Baker 1995). PI calves are consid-ered the most important source of infection, excreting high amounts of virus continuously during their life via nasal discharge, saliva, semen, urine, feces, tears and milk (Fray et al., 2000).

Shipping fever pneumonia is precipitated by stress-inducing con-ditions such as shipping, viral infec-tions, inhalation of diesel fumes and overcrowding (Frank, 1989) Mann-heimia haemolytica resides in small numbers in the upper respiratory tracts of cattle (Shoo and Wise-mens, 1990). Cells proliferate under stressful conditions and are aerolized in large numbers into lung alveoli, where they cause the disease (Frank and Briggs, 1992 and Gagea et al., 2006a).

Mycoplasma bovis is the most pathogenic mycoplama species in countries free of contagious bovine pleuropneumonia. Naturally occur-ring infectious pneumonias of clini-

cal significance usually have com-plex causes, after requiring the inter-action of mycoplasma with two or more organisms and with predispos-ing environmental factors (Jubb et al., 1993). Mycoplasma is a proven cause of pneumonia; septic arthritis and keratoconjunctivities (Pfützner and Sachse, 1996 and Kirby and Nicholas, 1996).

M. Bovis is percised as an emerging cause of mortality in feed-lot cattle associated with broncho-pneumonia and arthritis and the rela-tionship of this condition with BVDV infection as well as with bac-terial infection with Pasteurella mul-tocida. (Gagea et al., 2006a,b)

In Egypt, several studies have been implicated for the occurrence of deaths in imported cattle herds by Shehab et al. (2001) who related deaths to respiratory disorders due to mixed Mycoplasma bovis , Pas-tereulla multocida and streptococcal infection and Aly et al. (2003) who reported deaths due to outbreak of BVD viral infection either alone or mixed with other viruses in imported dairy herds quarantined at govern-mental quarantine of El-Beharia province.

T he objective of the present study was to investigate the

etiologic agents of mortalities in im-ported cattle herds from foreign


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countries to Egypt on the basis of virological, bacteriological, my-coplasmal and pathological as-pects. It is also a trial to identify the antibiogram of the isolates of bacterial pathogens.

MATERIAL AND METHODS

I. Animals:

T wo Frisian cattle herds were imported from Romania to

Egypt for slaughter purposes dur-ing November 2006 and March 2007 and quarantined at the gov-ernmental animal quarantine at El-Ein El-Sokhna (Suez province). The age of animals ranged from one to two years old.

a-Herd I: it consisted of 1354 male calves of which 113 calves were died within 10 days after arrival (the day of authors visit). The diseased animals in the herd suffered from respiratory manifestations (cough and dysp-nea), ptyalism, lacrimation, and mucopurulent nasal discharge, incoordination, recumbency and anorexia.

b-Herd II: It was 2205 male calves of which 226 calves died within two months after arrival (the day of authors visit). The diseased animals displayed the same clinical signs observed in herd I, beside profuse watery diarrhoea, abdominal respiration

and emaciation.

II. Samples: Internal organs: eleven dead

calves were necropized in the two herds and tissue specimens from the internal organs were se-lected for virological, bacterio-logical, mycoplasmal and histo-pathological examination.

Swabs: nasal, ocular and rec-tal swabs were obtained from 34 diseased calves for virological, bacteriological and mycoplasmal examination.

Serum and blood samples: serum samples and blood sam-ples on anticoagulant were col-lected from the same 34 diseased calves for serological diagnosis.

Tissue specimens were imme-diately immersed in 10% neutral buffered formaline solution mean-while the other samples were col-lected in an ice box and transferred directly to the laboratory for diag-nosis.

III. Virological examinations:

1-Agar gel precipitation test (AGPT): according to Hanel (1993)

Samples (buffy coat, nasal swabs, ocular swabs, rectal swabs and internal organs) were tested against standard reference positive hyperimmune sera, the agar was


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used in concentration of 1% in PBS and reaction was incubated for 24-48 h at 37°C in Co2 incuba-tor and then examined for the pres-ence of precipitin line.

2– Institute pourquier ELISA kits:

These kits were used for de-tection of NSP2-3 and Eo of the bo-vine virus diarrhoea/mucosal dis-ease virus (BVDV).

Assay procedure: Specific monoclonal antibodies directed to NSP2-3 and Eo are supplied and coated on the mi-croplate wells The samples to be tested were incubated in the wells with a buffer containing polyclonal anti-body directed specifically to BVDV, 50 µl of "sample incuba-tion buffer" per well + 50 µl of sample to be tested per well and 50 µl of positive and negative control antigens (100 µl per well). The upper left two wells act as negative control and the next two wells act as positive control.

The plate was incubated for 90 min. after covering with a lid. Af-ter 90 min. and after each step the plates were washed 3 times with washing buffer (if BVDV antigen were present in the analyzed sam-ples a polyclonal antibody anti-gen, monoclonal antibody com-

plex is formed).

After washing, a peroxidase con-jugated directed to polyclonal an-tibodies was incubated in wells for 30 min. 100 µl of diluted con-jugate (1/10 in the wash solution) in presence of the BVDV antigen, NSP 2-3 or Eo in the samples, the conjugate binds on the plate. After washing the enzyme sub-strate (TMB) was added to the conjugate 100 µl/well and incu-bated at room temperature for 20 min, away from light, 100 µl of stopping solution was added per well, the plates were shacked gently to homogenate the colored solution. The recording of the plate was observed at optical den-sity (OD) 450 nm wave length. The photometer must be first blanked on air.

Interpretation of the results: Calculate for each sample the S/P ratio (in %) S/P = OD of sample – OD of nega-tive control / OD of positive con-trol –OD of negative control x100

If sample S/P% < or = 25% are considered to be from animals that are not carrier to BVDV.

If sample S/P% is between 25%-30% are considered to be doubtful.

If sample S/P% = or > 30% are considered from animals carrier to the BVDV.


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3- Cell culture: A continuous cell line of

Madian Darby Bovine Kidney (MDBK) was used for trials for isolation and propagation of BVDV from (buffy coat, nasal swabs, rectal swabs and internal organs) which was positive in ELISA test and also used in virus neutralization test, the cell proved to be free from non-cytopathic st-rains of BVDV. Monolayer cell line was grown in Eagles MEM supplemented with 10% fetal calf serum. The cell was obtained from virology department, Animal Hea-lth Research Institute (AHRI).

The collected samples after preparation were subjected for vi-rus isolation via propagation on MDBK cell line according to the method described by Clark et al. (1984). Inoculated cells were incu-bated at 37°C and were examined daily for 5 days post incubation for three successive blind passages, CPE changes being to appear at the fourth passage.

4- Indirect immunofluorescent technique ( Indirect IFAT):

The (IFAT) was used on the inoculated cell culture with cyto-pathic effect (CPE) to identify the (cp BVDV), it also was used on inoculated cell culture without CPE to detect (ncp BVDV). The indirect IFAT was carried out ac-cording to OIE standers (1992).

5- Enzyme conjugate:

Anti-bovine fluorescence iso-thiocyanate conjugate was supplied by Sigma immunochemicals used in IFAT.

6- Reference positive immune sera: Standard reference positive

bovine hyper-immune serum of BVDV was supplied by virology department AHRI

7- Virus neutralization test:

It was conducted for detection of specific BVD-V neutralizing an-tibodies in cattle serum samples according to OIE (2004).

IV- Bacteriological examination: a- Cultural and biochemical

identification: A loopful of samples was sub-

cultured on brain heart infusion agar supplemented with 5% defeb-rinated sheep blood and MacCon-key agar. The plates were incu-bated at 36°C for 24 hs (Kodjo, et al. 1999). Bacterial identification was assessed by the observation of the colonial morphology, gram staining and biochemical identifi-cation including oxidase, catalase, urease testes, triple sugar iron agar, motility test and indol test (Atlas, 1997; Baily and Scott`s, 1998 and Toply and Wilson, 1998). b- Biotyping of isolated M.ha-

emolytica:


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Biotyping of isolated M.hae-molytica was applied depending on L-arabinose, trehalose, D-xylose, lactose and salicine fermentation tests (Biberstein et al., 1990). c- Virulence Studies of isolated

M.haemolytica: Eighty white mice were used

in 3 groups to estimate the viru-lence of local strains isolated from different samples by inoculation S/C with 0.2 ml M.haemolytica (3x108) according to Refai (1990).

d- Antimicrobial susceptibility test:

The susceptibilities of isolates to antimicrobial agents were deter-mined by using the disk diffusion method according to the NCCLS guidelines (2002). The antimicro-bial disks used are Ampicillin, Amikacin, Ceftiofur, Cephalo-ridine, Cephalexin, Donax, Eryth-romycin, Gentamicin, Norfloxacin, Oxytetracyclin, Pencillin G Strep-tomycin and Nalidixic acid. V- Mycoplasmal investigation: 1- Primary isolation of my-

coplasma and Acholoplasma were done using heart infusion broth and agar as described by Sabry and Ahmed (1975).

2- Differentiation between my-coplasma and acholoplasma was done by using digitonin test ac-cording to Freurdt (1983).

3- Biochemical identification: Glucose fermentation test: (Erno and Stiphovits, 1973):

The suspected cultures were inocu-lated into glucose medium and in-cubated at 37ºC. The change of color from red to yellow or orange was considered positive.

VI- Pathological examination: Representative tissue speci-

mens from lungs, livers, hearts, kidneys, spleen, mesenteric lymph nodes, rumen, reticulum, small and large intestines were fixed in 10% neutral buffer formalin solution for at least 24 h. They were routinely processed by standard paraffin em-bedding technique, sectioned at 4 µ and stained with: 1– Hematoxylin and Eosin (Ba-

ncroft et al., 1994) 2- Masson`s trichrome stain for de-

tection of fibrous connective tissue according to Clayden (1971).

RESULTS

Virological studies: Table (1) showed the results

of comparison between AGPT and ELISA test, where only 33 samples (13.4%) were positive to BVDV by using AGPT while 104 samples (42.3%) were positive to BVDV by using ELISA test.

The isolation of BVDV from samples represented in Table (2), gave positive results in ELISA test (104 samples), where 43 samples (41.3%) gave signs of CPE, the cy-topathic inducing viral agents were identified by IFAT, 37 out of 43 samples (35.5%) were identified as


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BVDV. (Fig.1). Table (3) illustrates the results

of BVDV specific antibodies in ap-parently healthy and diseased ani-mals in serum samples by using VNT, 13 serum samples (43.3%) of apparently healthy animals were positive in a titer (1/4-1/16) while the diseased ones showed that (70.6%) serum samples were posi-tive in a titer range (1/8-1/64).

Bacteriological studies: Occurrence of M.haemolytica

isolates in diseased and dead calves was illustrated in Table (4), where 75 M.haemolytica (41.2%) were isolated from samples of diseased and dead calves.

Sixty nine M.haemolytica were identified as biotype A and the rest (6) was identified as bio-type T. (Table 5)

Virulence test of 75 M.haem-olytica revealed that 67/69 mice were died within 12-24 hs (group 1) and the 5/6 died within 24-48hs and the control ones (5) still alive Table (6).

Antimicrobial susceptibility test revealed that M.heamolytica biotype A was highly sensitive to norfloxacin followed by ampicillin and erythromycin (65%, 50% and 40% respectively) and highly resis-tant to ceftiofur, nalidixic acid, gentamicine, cephaloridine, oxytet-racycline and cephalexin (90%, 80%, 50%, 50%, and 45% respec-tively). Table (7)

M.haemolytica biotype T was highly sensitive to norfloxacin and erythromycin (75%, 50% respec-tively) and highly resistance to cephalexin, nalidixic acid, donax, and oxytetracycline (87.5%, 87.5%, 50% and 50% respec-tively). Table (8)

Mycoplasmal studies: Primary isolation of my-

coplasma yielded a total of 18 iso-lates from lungs of diseased calves and their associated lymph nodes with an incidence of 81.8%. The recovery rate of mycoplasma from pneumonic lungs of dead calves was 90.9% (10 isolates out of 11 samples) while 8 isolates were ob-tained from 11 associated mediasti-nal lymph nodes with recovery rate 72.7%.

The recovery rate of my-coplasma digitonin positive from positive specimens isolates were 9 (90%) from lungs and 7 (87.5%) from lymph nodes; 7 isolates (87.5%) from trachea; 5 isolates (83.3%) from liver; 4 isolates (80%) from spleen and 5 isolates (83.3%) from kidney (Table 9). In diseased calves, mycoplasma digi-tonin positive from positive nasal and ocular isolates was 12 (70.6%) and 12 (75%) respectively (Table 10).

There was one isolate typed as Acholoplasma from organs of dead calves, five isolates from nasal swabs and four isolates from ocular


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swabs of diseased animals. All mycoplasma isolates were

proved to be M.bovis, where the iso-lates were positive to glucose fer-mentation, negative to arginine hy-drolysis and positive to film and spot-formation.

Gross pathology: The dead calves showed subcu-taneous hemorrhages on most of the serosal surfaces, the hemorrhagic zones ranged from minute petechiae to ecchymotic hemorrhages. The thoracic cavity showed blood tinged straw colored exudates and in most cases, adhesion of the parital surface of the pleura to the inner surface of the thoracic cavity was evident. The pleura was extremely thickened and fibrotic, sometimes petechial hemor-rhages were seen on its parital sur-faces.

The lungs were bilaterally enlarged, edematous and congested with the appearance of multiple hemorrhagic foci ranged from punc-tuate to large ecchymatic hemor-rhages. On cut section, whitish, grayish and /or reddish zones were observed (marbled appearance). Sometimes whitish creamy pus was oozed out from the pulmonary bron-chi and bronchioles. Some parts of the pulmonary tissue exhibited ir-regular abnormal enlargements, on palpation; excessive amounts of air were recognized inside it. In one dead calf, both lungs exhibited vari-

able sized abscesses in the pulmo-nary tissue. On cut section, creamy whitish pus oozed out. The mediasti-nal and tracheobronchial lymph nodes were enlarged, edematous and hemorrhagic.

The pericardium displayed min-ute petechial hemorrhages on its sur-face. The pericardial fat in some dead calves appeared pale, yellowish in color with gelatinous texture. The endocardium showed small focal ar-eas of hemorrhages on its surface. Sometimes the myocardial muscles were soft and flappy in consistency.

The livers in most dead calves showed numerous whitish foci 2-5mm in diameter on its capsular sur-face. On cut section, it extended in the liver tissue shortly beneath the capsule.

The kidneys were pale in color with the presence of multiple whitish to grayish foci on the renal capsule. The ruminal mucosa was moderately congested and edema-tous. Small pale slightly elevated foci were observed on the mucosal surface.

The small and large intestines showed patchy congestion and hem-orrhages on the serosal as well as on the mucosal surfaces. Multifocal sharply demarcated small erosions and /or ulcerations were noticed on the mucosa of the small intestine. The intestinal contents of both small and large intestines were watery and of greenish coloration. The mesen-


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teric lymph nodes were enlarged, edematous, and hemorrhagic.

Microscopic pathology: 1- Lung:

The pulmonary blood vessels were engorged with blood with the appearance of focal hemorrhages in the pulmonary parenchyma. Some alveolar lumina were massively dis-tended with faintly eosinophilic fi-briller network of fibrin which con-necting in between alveoli through pores of Kohn in the alveolar walls.

The fibrinous exudate was stud-ded with inflammatory cells in the form of histiocytes, lymphocytes and neutrophils (fibrinocellular pneumonia) (Fig. 2 A&B). Mean-while, other alveoli showed thick-ened interalveolar septa due to pro-liferation of pneumocyte type II cells and infiltration with mononu-clear inflammatory cells.

Some alveolar lumina showed presence of cellular exudates con-sisted of erythrocytes admixed with histiocytes, lymphocytes and neutro-phils. (Fig. 3A). Moreover, other al-veoli showed intralveolar aggrega-tions of spindle-shaped or oat-shaped leukocytes which oriented in a swirly lamellations (Fig3 B).

In few instances, clumped exfo-liated necrotic alveolar cells (strongly eosinophilic) were de-tected within the alveolar lumina ad-mixed with inflammatory cells (Fig.4A). The pulmonary tissue showed presence of multifocal zones

of coagulation necrosis which appre-ciated as circumscribed homogenous strongly eosinophilic area sur-rounded by histiocytes, lympho-cytes, plasmacytes and neutrophils (Fig.4B). Sometimes, oat-shaped leukocytes were evident at the vicin-ity of the necrotic zone. Moreover, coccoid bacteria were heavily colo-nized at the periphery of some ne-crotic foci (Fig. 5A).

The interlobular septa were thickened by fibrous connective tis-sue proliferation that was infiltrated with lymphocytes, histiocytes and neutrophils.

The bronchi and bronchioles displayed necrobiotic changes of their lining epithelium as well as mononuclear cell infiltration in the mucosal layers with the presence of exudates in their lumina composed of exfoliated epithelium and cellular debris (Fig., 5B). Emphysema of some pulmonary alveoli was evident in many instances (Fig. 5B).

In one dead calf, numerous small to large abscesses were recog-nized in the pulmonary parenchyma and replaced considerable parts of the pulmonary tissue. Each abscess composed of a central caseated or calcified core surrounded by heavy aggregations of neutrophils, histio-cytes and lymphocytes and bounded by fibrous connective tissue capsule. Thrombosis of some pulmonary blood vessels was observed associ-ated with degenerative changes of


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the tunica media (Fig. 6A). More-over, vasculitis with marked degen-erative and /or necrotic lesions of the endothelial layers was recog-nized in other blood vessels.

The pleura showed widely di-lated blood vessels, focal hemor-rhages and faintly eosinophilic fi-brinous exudates intermingled with inflammatory cells composed of histiocytes, lymphocytes and neu-trophis (Fig. 6B). Fibrous connec-tive tissue proliferation was evident at some parts of the pleura.

2- Liver: The hepatic tissue showed fo-

cal hemorrhages, disorganization of the hepatic cords, individualization of the hepatic cells and dilatation of the hepatic blood vessels and sinu-soids. Necrobiotic changes of the hepatic cells emphasized by clowdy swelling and vacuolar degeneration of the hepatocytes and multifocal hepatocellular coagulation necrosis (Fig.7A). The necrotic hepatocytes exhibited strongly homogenous eosinophil ic cytoplasm with karyolysis of the nuclei. Bacterial bacilli were detected in the lumina of some hepatic blood vessels and hepatic sinusoids and/or in between degenerated or necrotic hepatocytes (Fig. 7B &8A).

Increased fibrous connective tissue proliferation in the portal tri-ads was evident in many instances and infiltrated with histiocytes, lymphocytes and neutrophils. Hy-

perplastic proliferation of the bil-iary epithelium with the formation of more than one bile ductules and focal injury of endothelial cells of some hepatic blood vessels was ob-served associated with mild to mod-erate vasculitis (Fig.8B). Fibrous connective tissue proliferation in the Glisson`s capsule was detected in many instances.

3-Heart: There were multifocal hemor-

rhages in between cardiac myo-cytes. The pericardial sac showed faintly eosinophilic edema fluid ad-mixed with erythrocytes and mono-nuclear cells. Moreover, intermus-cular and perivascular edema were also evident.

Some cardiac myocytes re-vealed vacuolar degeneration em-phasized by intrasarcoplasmic vacu-oles of variable sizes (Fig.9A). Meanwhile, other cardiac myocytes exhibited necrotic lesions mani-fested by strongly eosinophilic sar-coplasm, loss of striation and karyolysis of the nuclei. In few in-stances, some cardiac myocytes ap-peared fragmented (Fig.9B). 4- Kidney:

The renal blood vessels were engorged with blood. The renal in-terstitial tissue showed focal hemor-rhages and mononuclear cell infil-tration represented by histiocytes, lymphocytes and plasmacytes.

Some renal corpuscles showed hypercellularity of the glomerular


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tufts due to proliferation and swell-ing of the endothelial and mesan-gial cells as well as due to neutro-philic and monocytic infiltration (prolife-rative glomerulonephritis) (Fig. 10A). The hypercellular glomerular tufts showed adhesion to the Bowman`s capsule and com-pletely obliterated the Bowman`s space with marked obsolescence of the glomerular tufts.

Meanwhile, other glomerular tufts showed marked thickening of the glomerular basement mem-brane with proliferation of the en-dothelial and mesangial cells as well as inflammatory cell infiltra-t ion (membranous glomeru-lonephritis) (Fig. 10B). The tubular epithelium showed various patho-logical enteties manifested by clowdy swelling and coagulation necrosis of the tubular epithelium with the presence of hyaline, epithelial or erythrocytic casts within the tubular lumina.

In one dead calf, chronic glomerulonephritis was recognized represented by extensive fibrous connective tissue proliferation in the renal interstitial tissue, peri-glomerular fibrosis and atrophied hypocellular glomerular tufts (Fig.11A). Moreover, dystrophic calcification emphasized by depo-sition of basophilic structurless materials in the interstitial tissue, in some glomerular tufts and in some tubules was recognized (Fig.11B).

5- Rumen and reticulum: The mucosa of both rumen

and reticulum exhibited group of cells showing degenerative and ne-crotic lesions in the stratum spino-sum and stratum granulosum (Fig. 12). The cells were swollen with karyopyknosis or karyolysis of the nuclei. Mononuclear cell infiltra-tion was seen in the lamina propria of both rumen and reticulum.

Sometimes the lamina epithe-lialis of both rumen and reticulum were heavily colonized by coccoid bacteria. 6- Small and large intestine:

The mucosal and submucosal blood vessels were engorged with blood. The intestinal mucosa showed coagulation necrosis of the surface enterocytes manifested by homogenous strongly eosinophilic cytoplasm with karyolysis of the nuclei (Fig.13A). Sometimes, the brush borders of the necrotic villi were heavily colonized with coc-coid bacteria (Fig.13B). The sur-face enterocytes may be exfoliated into the intestinal lumina leaving microerosions. (Fig.14A) or the en-tire mucosa may be completely ex-foliated into the lumina and ulcera-tive enteritis was observed (Fig. 14B). In few instances, coccoid bacteria were admixed with ne-crotic debris of the exfoliated sur-face enterocytes. The cryptal en-terocytes exhibited degeneration and necrotic changes and may be denuded into the cryptal lumina


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(Fig. 14A). The lamina propria showed edema with mild to heavy mononuclear and polymorphnu-clear inflammatory cell infiltra-tions.

The mucosa of large intestine showed extensive infiltration with inflammatory cells in the form of lymphocytes, histiocytes and neu-trophils. The intestinal crypts were either cystically dilated or showed hyperplasia of goblet cel ls (Fig.15A).

The submucosal blood vessels exhibited fibrinoid necrosis empha-sized by strongly eosinophilic ho-mogenous fibrinous deposits in the tunica intima associated with mononuclear and polymorphnu-clear leukocytic infiltration in the medial and adventitial layers (Fig.15B). Edema around some of the submucosal blood vessels was evident

The tunica muscularis were heavily infiltrated with mononu-clear and polymorphnuclear leuko-cytes. 7- Spleen:

In all dead calves, marked de-pletion of lymphoid cells in the white pulp of the spleen with ne-crosis of some cells in the germinal centers was observed. (Fig.16A). Moreover, necrosis of some lym-phoid cells in the splenic paren-chyma was evident in many in-s tances . Golden brown he-mosiderin pigment appeared to be

dispersed in the splenic paren-chyma. Splenic sinusoids were widely dilated and splenic trabecu-lea showed focal mononuclear cell infiltration.

8- Mesenteric lymph nodes:

Focal hemorrhages were ob-served in the parenchyma of the node and in some lymphoid folli-cles. The lymphoid follicles showed depletion of lymphoid cells with necrosis of some cells in the germinal centers leaving net-work of stromal cells.

Depletion of lymphoid cells was also evident in the cortical and paracortical zones as well as in the parenchyma of the lymph nodes. The lymphatic sinuses, subscapsu-lar, peritrabecular and medullary sinuses were dilated and displayed presence of cellular exudates com-posed of erythrocytes admixed with histiocytes and lymphocytes (fig. 16B). Meanwhile other dilated lymphatic sinuses contained exag-gerations of histiocytes within its lumina (sinus histiocytosis).

The mesenteric lymph nodes in one dead calf showed chronic lymphadenitis manifested by fi-brous connective tissue prolifera-tion in the parenchyma particularly around the lymphoid follicles (Fig.17A). It was confirmed by us-ing Masson`s trichrome stain (stained green) (Fig.17B).


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Table (1): Results of comparison between AGPT and ELISA test for detection of BVDV an-tigen in the diseased and dead animals.

State of animals Sample tested No. of samples +ve by AGPT

No. of isolates +ve by ELISA test

diseased

Type No. Buffy coat 34 6 22

Nasal swabs 34 5 20 Ocular swabs 34 - - Rectal swabs 34 2 8

Dead

lung 11 - - trachea 11 - - Liver 11 3 8

Spleen 11 2 6 Mesentric L. N 11 3 9

Kidney 11 1 4 Heart 11 1 3

Intestine 11 4 8 Rumen 11 3 8

Reticulum 11 3 8 Total No. 246 33 104

Overall % 13.4 42.3

Table (2): Results of isolation and identification of BVDV from diseased and dead animals.

State of animals

Sample tested No. of samples with CPFT

Virus identifi-cation by IFAT

Type No.

Diseased calves

Buffy coat 22 6 6 Nasal swabs 20 4 3 Rectal swabs 8 5 3

Dead calves

Liver 8 4 3 Spleen 6 3 2

Mesentric L. N 9 4 4 Kidney 4 2 2 Heart 3 2 1

Intestine 8 5 5 Rumen 8 4 4

Reticulum 8 4 4 Total 104 43 37

Overall % 41.3 35.5


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Table (3): Result of BVDV specific antibodies in apparently healthy and diseased calves in serum samples by VNT.

State of ani-mals

No. of tested sera

No. of +ve % of +ve Average titer

Apparently healthy 30 13 43.3 1/4-1/16

Diseased 34 24 70.6 1/8-1/64 Total No. 64 37 57.8

Table (4): Occurrence of M. haemolytica isolates in diseased and dead calves.

State of ani-mals

Type of sam-ples

Total num-ber of sam-

ples

Isolated M.haemolytica

Diseased calves (34)

No. % Nasal swab 34 27 79.4 Ocular swab 34 0 0 Rectal swab 34 0 0

Dead calves (11)

lung 11 10 90.9 liver 11 8 72.7

spleen 11 9 81.01 kidney 11 8 72.7

Gall bladder 11 0 0 Lymph nodes

11 11 100

Intestine 11 2 10.18 Total 179 75 41.2

Table (5): Biotyping of isolated M. haemolytica

No. of total M. haemo-

lytica isolates

Biotype A Biotype T

No. % No. %

75 69 92 6 8


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Table (6): Results of virulence test of isolated M. haemolytica

M.haemolytica biotype

Total No. of mice

Mortality Time of death No %

Biotype A 69 67 97.1 12-24 Biotype T 6 5 83.3 24-48 Control 5 0 0 -

Total 80 72 90 12-48

Table (7): Antimicrobial susceptibility tests of Biotype A (20 isolates as a representative isolates.

Antimicrobial disk

Sensitive

Resistant

mild moder-ate

strong

No. % No. % No. % No. % Ampicillin 10µg 6 30 2 10 10 50 2 10 Amikacin 30 µg 4 20 4 20 6 30 6 30

Ceftiofur 30 µg 2 10 0 0 2 10 18 90

Cephaloridine 30 µg 1 5 3 15 6 30 10 50

Cephalexin 30 µg 2 10 1 5 8 40 9 45

Donax 5 µg 2 10 4 20 5 25 9 45

Erythromycin 15 µg 3 15 5 25 8 40 4 20 Gentamicin 120 µg 2 10 2 10 4 20 12 60

Norfloxacin 10 µg 1 5 3 15 13 65 3 15

oxytetracyclin 30 µg 2 10 5 25 3 15 10 50

Pencillin G 10 units 1 5 6 30 5 25 8 40

Streptomycin 10 µg 3 15 6 30 2 10 9 45 Nalidixic acid 30 µg 1 5 1 5 2 10 16 80


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Table (8): Antimicrobial susceptibility tests of Biotype T (8 isolates).

Antimicrobial disk

Sensitive Resistant

mild moderate strong No. % No. % No. % No. %

Ampicillin 10µg 4 50 2 25 0 0 2 25 Amikacin 30 µg 3 37.5 1 12.5 3 37.5 1 12.5 Ceftiofur 30 µg 2 25 1 12.5 0 0 3 37.5 Cephalo-

ridine 30 µg 2 25 0 0 2 25 4 50

Cephalexin 30 µg 1 12.5 0 0 0 0 7 87.5 Donax 5 µg 2 25 1 12.5 0 0 4 50

Erythromycin 15 µg 1 12.5 1 12.5 4 50 2 25 Gentamicin 120 µg 0 0 2 25 2 25 4 50 Norfloxacin 10 µg 0 0 1 12.5 6 75 1 12.5

oxytetracyclin 30 µg 1 12.5 0 0 3 37.5 4 50 Pencillin G 10 units 1 12.5 1 12.5 3 37.5 3 37.5

Streptomycin 10 µg 1 12.5 3 37.5 3 37.5 1 12.5 Nalidixic acid 30 µg 1 12.5 0 0 0 0 7 87.5

Table (9): Recovery rate of mycoplasma and Acholoplasma from different organs col-lected from dead animals

Type of sample

No. of samples

Positive isolates Mycoplasma Acholoplasma No. % No. %* No. %*

Lung 11 10 90.9 9 90 1 10

Lymph nodes

11 8 72.7 7 87.5 1 12.5

Trachea 11 8 72.7 7 87.5 1 12.5

Liver 11 6 54.5 5 83.3 1 16.7

Spleen 11 5 45.5 4 80 1 20

kidney 11 6 54.5 5 83.3 1 16.7

Table (10): Results of recovery rate of mycoplasma from examined samples of living diseased animals and biochemical characterization of the obtained isolates.

Type of sample

No. of samples

Positive isolates Mycoplasma Acholoplasma No. % No. %* No. %*

Nasal swabs 34 17 50 12 70.6 5 29.4

Ocular swabs 34 16 47 12 75 4 25

* % was calculated according to number of positive samples.


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Fig. (1): Normal MDBK non infected cells (A) and infected MDBK cells (notice specific intra-cytoplasmic fluorescent granules) (B).

Fig.(2 A & B): Lung of a dead calf illustrating fibrinous pneumonia, the alveolar lumina were massively distended with fibrinous exudate admixed with leukocytes (A & B. H & E x 400).


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Fig. (3 A&B): Lung of a dead calf showing: A: Cellular exudate in the alveolar lumina. B: Spindle or oat-shaped leukocytes in the alveolar lumina which orientd in the form of a swirly lamellations.(A&B. H & E. A x 200 B x 400)

Fig. (4 A&B): Lung of a dead calf illustrating A: Strongly eosinophlic clumped exfoliated necrotic ma-terial in the alveolar spaces admixed with inflammatory cells. B: Circumscribed zone of coagu-lation necrosis (pulmonary sequestrum) bounded by aggregations of inflammatory cells (A&B. H&E A:x 100, B :x 200).


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Fig: (5 A&B): Lung of a dead calf revealing: A: Heavy colonization of coccoid bacteria at the vicinity of the necrotic tissue. B: Degenerative and necrotic lesions of the bronchiolar epithelium with exfoliation into the lumina and emphysema of the adjacent alveoli. (A & B. H&E x 100)

Fig. (6 A & B): Lung of a dead calf showing: A: Thrombosis and degeneration of the tunica media of one of the pulmonary blood vessels. B: Fibrinouspleuritis:, the pleura showed deposition of fibrinous exudate admixed with inflammatory cells (A&B. H &E A x 200, B x 100)


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Fig: (7 A&B): Liver of a dead calf depicating: A: Focal hepatocellular coagulation necrosis. B: Widely dilated hepatic blood vessels with bacterial bacilli in their lumina (A & B. H & E x 400)

Fig. (8 A&B): Liver of a dead calf revealing: A: Bacterial bacilli in between necrotic hepatocytes. B: Intimal injury of the wall of one of the hepatic blood vessels. (A & B. H & E x 400).


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Fig: (9 A&B): Heart of a dead calf illustrating: A: Vacuolar degeneration of the cardiac myocytes. B: Necrosis and fragmentation of the cardiac myocytes. (A & B, H&E x 400)

Fig. (10 A & B): Kidney of a dead calf illustrating: A: Hypercellularity of the glomeruler tufts with ad-hesion to the Bowman`s capsule (proliferative glomerulonephritis). B: Membranous glomeru-lonephritis; the basement membrane of the glomerular tufts appeared thickened with mono-nuclear inflammatory cell infiltration. (A & B, H & E, A x 200, B x 400)


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Fig: (11 A&B): Kidney of a dead calf showing: A: Chronic glomerulonephritis represented by periglomerular and interstitial fibrosis, hypocellular atrophied glomerular tufts and plas-macytic, lymphocytic infiltration of the renal tissue. B: Dystrophic calcification of the intersti-tial tissue and renal tubules. (A&B. H&E, A x 400, B x 200)

Fig. (12): Rumen of a dead calf showing degenerative and necrotic lesions in the cells of the stratum spinosum of the ruminal mucosa. (H &E x 400)


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Fig: (13 A & B): Small intestine of a dead calf illustrating: A: Necrosis of both surface and cryptal en-terocytes. B: Coagulation necrosis of the surface enterocytes and the brush border of the ne-crotic villi was heavily colonized with coccoid bacteria. (A&B H&E A x100, B x200)

Fig. (14 A & B): Small intestine of a dead calf demonstrating: A: Microerosions of the intestinal mu-cosa. B: Ulceration of the intestinal mucosa (A & B. H & E x 200).


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Fig: (15 A & B): Large intestine of a dead calf revealing: A: Hyperplasia of goblet cells in the intestinal glands. B: Fibrinoid necrosis of one of the submucosal blood vessels associated with vasculitis and perivascular odema. (A & B. H & E x 200)

Fig. (16 A & B): Spleen of a dead calf showing: A: Depletion of lymphoid cells in the white pulp of the spleen. B: Mesenteric lymph node of a dead calf illustrating dilated lymphatic sinuses that con-tained erythrocytes admixed with histiocytes and lymphocytes (A & B. H & E x 200)


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Fig. (17 A & B): Mesentric lymph node of a dead calf demonstrating chronic lymphadenitis: A: Extensive fibrous connective tissue proliferation in the lymph node parenchyma around the lymphoid follicle. B: The proliferated fibrous connective tissue stained green (Masson`s trichrome stain x100)

DISCUSSION

I n the present study mortalities in two imported Frisian cattle

herds were documented within 60 days after arrival during 2006 and 2007 in governmental quarantine at El-Sokhna (Suez governorate). Most deaths were mainly related to respiratory diseases and digestive tract disorders. There are many factors that expected to compro-mise the immune function in ani-mals after arrival encompassed change in animal health, manage-ment, environmental conditions, diets and commingling of animals

as well as transportation stress. (Loneragan et al., 2001) In Egypt, deaths in imported quar-antined animals were previously recorded in fattening veal calves by Shehab et al., (2001) who attrib-uted deaths to mycoplasmal pneu-monia and Pasteurella multocida and Aly et al., (2003) who related deaths in 3 cattle herds to BVD vi-ral infection either alone or mixed with other viruses.

The goal of this study is to isolate and identify the causative agents of these high mortalities and study their histopathological le-


103

sions in dead animals. Amiridis et al. (2004) pointed out

that BVB viral infection was responsi-ble for severe deaths (22.85%) in 70 im-ported herds of Holstein heifers within six months after arrival.

The immunesuppressive effect of acute BVDV infections can enhance the clinical disease of other pathogens and play an important role in multiple infec-tious diseases (Potgieter, 1997). The immunesuppressive effect of BVDV mainly due to strong affinity of the virus for immuno-competent cells which may be destroyed or functionally impaired (Potgieter, 1995).

BVDV depresses immunoglobu-lin and interferon production, reduces response of lymphocytes to mitogens and impairs monocyte chemotaxis. This depression of cell mediated immunity is accompanied by reduced humeral im-mune response. (Jubb et al., 1993)

In the present study, AGPT was used as a simple and rapid method for BVDV or antigen detection in collected samples (buffy coat, nasal, ocular and rectal swabs and internal organs) which was reported by (Hanel, 1993; Hosny et al., 1996 and Nahed et al., 2004). Only 33 (13.4%) samples out of 246 samples were positive. Results of AGPT are low if compared with other tests due to the low sensitivity of the test or time of sample collection. Commercial Pourquier ELISA kits (antigen capture ELISA) gave rapid and accurate detec-

tion of BVDV antigen in the same origi-nal samples, where 104 (42.3%) out of 246 samples were positive.

An antigen capture ELISA was de-veloped and proved its specificity, sen-sitivity and accuracy (Gottschalk et al. 1992). This coincidence with our view results of commercial ELISA kits for detection of BVDV in the field samples. Also, Donis (1995) reported that anti-gen detection ELISA for BVDV was useful mainly in confirming enteric, res-piratory and reproductive diseases. Many authors described the importance of ELISA in testing program of animals to control BVDV (Sandvik et al., 1997 and Ferrari et al., 1999).

ELISA test was improved by the use of monoclonal antibodies which were specific to BVD viral proteins and we could detect most if not all BVDV strains (Sandvik, 1999). In our study we use kits with specific monoclonal antibodies directed to NSP2-3 (NSP-3 associated with lytic activity of the cy-topathic strain) which increased the ap-plication of ELISA for detection of BVDV strains and offered sensitivity equivalent or higher than virus isolation, this also recorded by Ferrari et al. (1999); Cavirani et al. (2000) and OIE (2000). Also, Aymen (2002) proved that commercial ELISA kits are valu-able in antigen detection.

MDBK cell line is considered the most common cell culture system for virus isolation and propagation (Allam, 2000)


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In the present study, MDBK cells were used for three successive blind passages for samples positive by ELISA, out of 104 samples, 43 gave signs of CPE for BVDV on the cell. These samples were then subjected for i den t i f i c a t i on by i nd i r ec t im-munoflourescent technique (IFAT) to detect non-cytopathic BVDV, where all gave negative results. Edwards (1990) and BrocK (1991) reported that the conventional diagnosis of Pestivirus is based on direct detection of the virus in the clinical samples by using cell cul-t u r e me thod fo l l owed by im-munoflourescence. Also, Haines et al. (1992); Abd-El Rahim and Grunder (1996), reported that accurate diagnosis of BVDV infection depend upon isolat-ing the virus from blood or nasal swabs or tissue samples from affected animals in diagnostic laboratory which agrees with our results.

The virus multiplication was de-tected by immunoflourescent technique that revealed diffuse or granular intra-cytplasmic fluorescen in inf-ected cells. The same results were reported by (Liess et al., 1974) who detected BVDV in organs of 11 from 14 dead cattle by (IFAT), also Munoz et al., (1996); Tsuboi and Imada (1999) and Zabal et al. (2000) used IFAT for identification of BVDV cell culture. The samples positive by ELISA kits and did not iso-late or identify by IFAT may be due to the BVDV present in those negative samples FA complexes with antibodies rendered it non infectious for cell cul-

tures (Palfiet et al., 1993). Improper handling or storage of the samples, in-stability BVDV, also ELISA can detect both BVDV and antigen. ELISA has ad-vantage of speed handling large number and overcome low sensitivity other tests (Aymen, 2002). Serological examina-tion of serum samples applied for de-tecting antibodies specific to BVDV is a useful tool for herd screening and BVDV prevalence and monitoring BVDV free herd status (Houe et al., 1995).

VNT was carried out for detection of specific neutralizing antibodies for BVDV in both diseased and apparently healthy animals. The results showed that 13 (43.3%) serum samples out of 30 apparently healthy animals were positive and 24 (70.6%) out 34 diseased animals were positive. Antibody tests were useful in assessing the status of animal groups as a part of disease con-trol. VNT is the most common serologic test used as a reference method for BVDV tests (OIE, 2004).

M. haemolytica is a gram negative coccobacillus usually found as a com-mensal in the nasal passages of cattle (Abdullah et al., 1992). There are many predisposing factors such as, transporta-tion, overcrowding and viral infection which may cause the M.haemolytica to shift from being commensal to pathogen (Confer et al., 1995). When a viral in-fection exists, the mucosal lining is de-graded and the infection can move to the lower respiratory tract. Remaining defenses fail to resist infection because


105

of virulence factors of the bacteria. M.haemolytica possesses various

putative virulence determinants (Hi-ghlander, 2001) including a transfer-ring receptors (Ogunnariwo and Schryver, 1990 and Yu et al., 1992); leukotoxin (Kaehler et al., 1980 and Davis et al., 1997) which are specific for ruminant transferin (Yu et al. 1992) and lymphoid cells (Berggen et al., 1981; Clinkeubeard et al., 1989; Davis and Lee, 2004 and Atapattu and Czu-prynski, 2005). M. haemolytica also, produce toxins which inhibit the pro-duction of polymorphnuclear neutro-philic granulocyte (PMN), and chemo-tactic factors which kill bovines PMNs and macrophages (Morck et al., 1987 and Davis et al., 2001). However, cap-sular polysaccharides and lipopolysac-charides play a relative important role in the disease process. (Confer and Simons 1986; Adlam, 1989; Slocombe et al. 1990 and Davis et al. 1997).

In the present study, 75 M.haem-olytica (42.3%) were isolated from dead and diseased calves. The high percent-age of M.haemolytica may be attributed to more than one stress factors as ship-ping, viral infection (BVDV) and my-coplasma infection (Confer et al. 1995 and Highlander, 2001).

M. haemolytica were isolated from most specimens, this may be due to bac-terimia (Baily and Scott`s, 1998).

Two biochemical types of M.ha-emolytica are recognized and are de-

signed Biotype A and T, the letter stand for arabinose or trehalose fermentation (Adlam, 1989 and Muttlers et al., 1989).

Sixty nine (92%) of the isolated M. hemolytica were biotyped as biotype A and 6 M. haemolytica (8%) were bio-typed as biotype T. These results re-vealed that M. haemolytica biotype A was the most frequently associated with shipping fever, a disease of beef cattle which characterized by fibrinous pleu-ropneumonia (Abdullah et al. 1992; Stevens and Czuprynski 1996; Reggie et al. 2001; Linda and Reggio 2002;Ewer et al. 2004 and Ilhan and Keles, 2007).

On virulence test, 90% of inocu-lated mice were died within 12-48h. About 97.1% of mice inoculated with biotype A were died within 12-24hs while about 83.3% of mice inoculated with biotype T were died within 24-48 hs. These results were nearly similar to that reported by Omnia (2006) and Odugbo et al. (2004) who reported the virulence of M.haemolyt-ica biotype A on sheep.

On antimicrobial susceptibility tests, most of M. haemolytica were sen-sitive to norfloxacin, ampicillin and erythromycin. These results were agreed with Esaki et al. (2005) and Catry et al. (2007), they were resistant to ceftio-fur, nalidixic acid, gentamicine, cephal-ridine, oxytetracycline, cephalexin and donax. These results were nearly similar


106

to that reported by Esaki et al. (2005) and Catry et al. (2005), and disagree with Mevius and Hartman (2000) and Berge et al. (2006), who recorded that M. haemolytica isolated from calves, sheep and goat were sensitive to ceftio-fur, gentamicine and oxytetracycline. These results revealed that virulence of the isolated biotypes were high and treatment of M. haemolytica for respira-tory tract disease is complicated by an-timicrobial resistance. These may be at-tributed to the misused of antimicrobial drugs before laboratory diagnosis and applying of susceptibility tests, where the two herds were medicated by differ-ent antimicrobial agents before labora-tory diagnosis.

In a survey conducted by the Of-fice Internationals de Epizooties (OIE), M.bovis was a major component of calf pneumonia complex, with the isolation rates of 23.35% (Nicholas et al., 2000).

The situation of mycoplasma, a se-ries of experiments designed by Hough-ton and Gourloy (1985), proved that there is synergism between pasteurella and Mycoplasma bovis in calf pneumo-nia. Also Abo El– Leil (1992) reported that in the extensive pleurisy, pasteurel-losis and/or mycoplasmosis could be suspected. Moreover, Gagae et al. (2006a) reported the pathogens associ-ated with mortality or severe morbidity for feedlot calves were BVDV, M.bovis and M.haemolytica.

In the present study, M.bovis was isolated from 9 (90%) from lungs and 7

(87.5%) from lymph nodes; 7 isolates (87.5%) from trachea; 5 isolates (83.3%) from liver; 4 isolates (80%) from spleen and 5 isolates (83.3%) from kidney. In diseased calves, M. bovis was isolated from nasal swabs 12 (70.6%) and ocular isolates 12 (75%).

These results were nearly similar to that reported by Gagea et al. (2006 a, b) who isolated M.bovis in a rate of 82% and is agree with Tenk et al. (2004) who screened 34 cattle for the presence of M.bovis, here it was isolated in a rate of 25.2%. The difference in the rate of isolation may be attributed to the number of examined samples.

At necropsy, the dead calves dis-played fibrinointerstitial bronchopneu-monia with pleuritis and adhesion of the parital surface of the pleura to the inner surface of the thoracic cavity. These morphological features may be related to severe toxemia incited by the infec-tion with M.haemolytica as well as to mycoplasmal infection wh-ich brings about fibrinous bronchopneumonia and fibrinous pleuritis as mentioned by Gagea et al. (2006 a & b) and MacGavin and Zachary (2007).

Moreover, these results are consis-tently similar to Donkergoed et al. (1993) who isolated M.haemolytica from pneumonic dead calves in 73% and Gagea et al. (2006a,b) who re-corded the common causes of pneumo-nia and death in imported beef feedlot calves within 2 months after arrival were M.haemolytica (27%) and M.bovis


107

(82%) and M.arginini in (72%). Histologically, the lung tissue ex-

hibited inflammatory exudates within the alveolar lumina, either fibrinous, fibrinocellular or cellular which may be admixed with cellular debris. Jubb et al. (1993) suggested that, the pro-duction of profuse fibrinous exudate correlates with a fulminating pulmo-nary inflammation and usually develop in cattle that have been recently stressed by transportation. Moreover, the detection of fibrinocellular exudate (fibrin admixed with histiocytes, neu-trophils and plasma cells) in other al-veolar lumina as well as in the pleural sac was attributed by (MacGavin and Zachary, 2007) to increased perme-ability of the blood-air barrier, in-creased procoagulant activity and di-minished profibrinolytic activity of the lung, furthermore, fibrin is chemotac-tic for leukocytes particularly neutro-phils.

The tendency of leukocytic ag-gregates (elongated cells with spindle shape nuclei or oat shaped cells) in the alveolar lumina is considered one of the pathognomic lesions of pneumonic pastereullosis and attributed to the tox-ins from gram –ve bacteria as de-scribed by Jubb et al., 1993; Jones et al. 1997; MacGavin et al., 2001 and Blowey and Weaver, 2003. Mean-while MacGavin and Zachary, 2007 suggested that oat-shaped leukocytes were degenerated neutrophils admixed

with alveolar macrophages. The detection of numerous co-

agulative pulmonary parenchymal ne-crotic zone bounded by leukocytic ag-gregates (pulmonary sequestra) is a true pathologic entity incited by the endotoxins as well as by leukotoxin of M.haemolytica that specifically de-stroy ruminants leukocytes (polymo-rphes, lymphocytes and histiocytes) at the site of infection and impairing im-mune response. (Bottone, 1998)

Another reason for extensive ne-crosis of lung tissue was related to the secretion of maximum amounts of cy-tokines by alveolar macrophages such as TNFα, interleukin I, and interleukin 8 with potent neutrophil chemotactic activities that contribute to injury and necrosis of the pulmonary tissue (Malazdrewish et al., 2001 and MacGavin and Zachary, 2007). Fur-thermore, vascutitis and thrombosis of the blood vessels incited by mycoplas-mal infection may responsible for large pulmonary sequestra due to pul-monary infarction. (Shahriar et al., 2002 and Gagea et al., 2006a)

Moreover, bronchitis, bronchioli-tis, pleuritis, thickened interlobular septa as well as thickened alveolar walls (interstitial pneumonia) were re-markable findings due to mycoplasmal infection which brings about immune suppression, ciliary dysfunction and unregulated production of TNFα. (MacGavin et al., 2001 and Shahr-


108

iar et al., 2002). The appearance of multiple in one

dead calf abscesses in the pulmonary tissue is a direct consequence of impair-ment of bacterial clearance by lysis of pulmonary macrophages and infiltrating leukocytes by M. haemolytica leuko-toxin as hypothesized by Bottone (1998). On the other side, Adegboye et al. (1995) regarded mycoplasma as one of the causative agents of bovine bron-chopneumonia with abscessation. Fibrinous pleuritis with adhesion to the thoracic cavity occurs as extension of pneumonic mannheimiosis or mycoplas-mosis as mentioned by Abo-El Leil (1992) and MacGavin and Zachary (2007). Many investigations stated that BVDV may infect lung tissue as it con-tributes to the pathogenesis of respira-tory diseases through immunosuppres-sion and acts synergistically with other respiratory pathogens to facilitate bacte-rial colonization and gave rise to pneu-monia. (Jubb et al., 1993; Hosny et al., 1996; Brodersen and Kelling, 1998; Aly et al., 2003 and Loneragan et al., 2005).

Reggiardo and Kaeberle (1981); Haines et al. (2004) and Gagea et al. (2006b) pointed out that BVDV infec-tion interfered with normal clearance mechanisms due to leucopenia since, animals with deficient humeral and cell mediated immunity die due to impair-ment of polymorphnuclear cell func-tions.

The liver in most cases exhibited hemorrhages, hepatocellular degenera-

tion and necrosis and vasculitis. These entities may be attributed to toxemia produced by M.haemolytica (Jones et al., 1997). Furthermore, mycoplasma infection brings about necrotic foci in the hepatic parenchyma due to thrombo-sis of the hepatic blood vessels.

Bacterial colonization was evident in the hepatic blood vessels and sinu-soids and in between degenerated hepa-tocytes. A picture which related to im-munosuppressive effect of BVDV which lead to ascending of bacteria from disrupted gastrointestinal tracts as reported by (David et al., 1994; Piero et al., 1997 and Ibtisam et al., 2000).

The heart muscles of dead calves displayed focal hemorrhages and fla-ppyness of cardiac-myocytes which ap-preciated microscopically as vaculations and necrosis of the cardiac myocytes. Similar findings were previously re-corded by David et al. (1994), and they accentuates the hypothesis of Piero et al. (1997) and Haines et al. (2004) who detected BVD viral antigens in pukinji fibers, ventricular muscles and medium sized arteries and suggested that these lesions may promote arrythmogenesis.

The kidneys revealed variety of pathological lesions emphasized by pro-liferative and membranous glomerulo-nephritis, necrosis of the tubular epithe-lium and plasmacytic, lymphocytic in-terstitial infiltration.

The emphysis of proliferative and membranous glomerulo-nephritis which represented the anatomic forms of im-munecomplex glomerulonephritis in-


109

cited by BVDV infection as hypothe-sized by Jubb et al. (1993) and MacGavin et al. (2001) who mentioned that BVDV infection brings about in-crease in the mesangial cells and in-criminated this infection as one of the causative agents of immunecomplex glomerulonephritis. Owing to the fact that BVDV induces a complex response by activating cells mediating the cellular and humeral im-mune response where antigen antibody complex formed in the circulation trapped in the glomeruli and induce glomerular damages (MacGvin et al., 2001 and Kumar et al., 2005).

Jubb et al. (1993) mentioned that the renal interstitial leukocytic infiltra-tion is a sequence of glomerular lesions. Moreover, David et al. (1994); Ibtisam et al. (2000) and Aly et al. (2003) re-ported the same lesions in kidneys of dead calves infected with BVDV. In one dead calf, the renal cortex displayed periglomerular and interstitial fibrosis and atrophied hypocellular glomerular tufts. These histological features repre-sented the signs of chronic glomeru-lonephritis as described by Jubb et al. (1993) and MacGavin et al. (2001). Dystrophic calcification was detected at some parts of the renal cortex. MacGavin et al. (2001) suggested that calcification of fibrotic kidney occurred due to alterations in calcium phosphorus metabolism associated with chronic re-nal failure.

The detection of numerous foci of

degenerated or necrotic epithelial cells in the stratum spinosum in the ruminal and/or reticular mucosa was recognized in dead calves which may prorgress to erosion and/or ulceration, Since BVDV targets the stratified squamous epithe-lium of the upper alimintary tracts. (David et al., 1994, Bordersen and Kelling, 1998; Ibtisam et al., 2000; Aly et al., 2003 and Amiridis et al. 2004). The intestinal tracts of dead calves exhibited severe congestion on both serosal and mucosal surfaces with numerous erosive and /or ulcerative lesions. These pictures were reported by many authors (Ibtisam et al., 2000 and Aly et al., 2003).

Histologically, there were various alterations manifested by necrotizing enteritis, inflammatory cell infiltration and fibrinoid necrosis and vasculitis of some submucosal blood vessels. MacGavin and Zachary (2007) ex-plored out that BVDV gain entrance to the intestinal mucosa through M cells (mem-branous or micro-folded surface cells) which serves as a portal of entry of some pathogens.

The necrotic lesions of the surface and cryptal enterocytes with exfoliation into the intestinal and cryptal lumina were explained by Mac-Gavin et al. (2001) who pointed out that necrotizing processes initiated at the gut associated lymphoid tissue with extension to the over lining epithelium.

These lesions were in attribute to potentiation of BVDV to the epithelial linings of the alimintary tracts as de-


110

scribed by Jubb et al. (1993); David et al. (1994); Brodersen and Kelling (1998); Ibtisam et al. (2000) and Aly et al. (2003).

Extensive damage of the cryptal epithelium leads to collapse of the lam-ina propria with subsequent ulceration which considered one of the diagnostic lesions of BVDV infection (Ibtisam et al., 2000 and Aly et al., 2003 and MacGavin and Zachary, 2007).

Hyperplasia of globlet cells was evi-dent in some intestinal glands which represented a feature of inflammatory conditions of the intestine as mentioned by Jones et al. (1997).

One of the most important micro-scopic lesion in the submucosal blood vessels was fibrinoid necrosis and vas-culitis which occurred due to increased vascular permeability that lead to insu-dation of fibrinogen into the subendo-thelial area and initiates inflammatory condition as suggested by Kumar et al. (2005). A finding which accentuates the hypophesis of Jubb et al. (1993), who mentioned that BVDV infected endo-thelial cells of intestinal blood vessels and brings about vasculitis.

One of the pathological hall marks in the spleen and mesenteric lymph nodes was a prominent lymphofollicular depletion as well as depletion of lym-phoid cells in the cortical and paracorti-cal zones. Since BVDV targets lympho-cytes and brought about necrosis and destruction of lymphoid cells in the lymphoid follicles and many studies re-corded perturbations of the immune sys-

tem by BVDV (Bordersen and Kelling, 1998; Ibtisam et al., 2000 and Aly et al., 2003)

Moreover, the mesenteric lymph nodes showed widely dilated lymphatic sinuses with the presence of erythro-cytes admixed with histiocytes and lym-phocytes. Jubb et al. (1993) mentioned that erythrocytes delivered to lymphatic sinuses in animals suffered from toxe-mic conditions. Meanwhile sinus histio-cytosis was evidenced in some mesen-teric lymph nodes. This specific entity indicate antigenic stimulation of the node as mentioned by Jones et al. (1997) and consistently correlated with Kumar et al. (2005) who speculated that sinus histiocytosis occurred in lymph nodes with continuated destruc-tion of its cells. Chronic lymphadenitis which detected in one dead calf repre-sented the end stage of various inflam-matory conditions of the node as sug-gested by (MacGavin et al., 2001).

CONCLUSION

M ortalities in imported calves may be attributed to mixed infection

of viral, mycoplasmal and bacterial in-fection (M.haemolytica).

Fibrinous pleuropneumonia was ob-served in this study, which considered as a complex disease observed in im-ported animals leading to high mortali-ties.

Mycoplasma bovis and M. haemo-lytica with BVDV virus as immunosup-pressive virus were considered as a


111

complicated infection in imported calves leading to fibrinous pleuropneu-monia.

Therefore the purchased stocks must be screened and tested for virus, my-coplasma as well as bacteria and in-fected animals should be identified.

It so important to pay attention to the role of BVDV in imported calves as raising the possibility of existence of PI animals. Those animals may cause ex-tensive shedding of the virus in the population and resulted in severe out-breaks with high mortalities. So, ani-mals imported to Egypt should be free from BVDV infection, PI animals must be detected and removed by applying control program to all imported herds.

Antimicrobial susceptibility tests should be applied directly after labora-tory diagnosis of M.haemolytica to avoid the misused of antimicrobial drugs and release of high multidrug re-sistant strains of M.haemolytica.

Environmental stress factors must be avoided by good ventilation and feeding, and over crowding during transportation.

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