DIARRHEA SYNDROME CAUSED BY CAMPYLOBACTER JEJUNI IN CALVES

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

DIARRHEA SYNDROME CAUSED BY CAMPYLOBACTER JEJUNI IN CALVES

HALA, M. ISMAIL¹ AND SHEREEN S. MOUSTAFA²

¹ Pathology Unit, (Animal Health Research Institute, Mansoura Lab), Agriculture Research Center (ARC).

² Bacteriology Unit, (Animal Health Research Institute, Mansoura Lab), Agriculture Research Center (ARC).

Received:20 April 2021;     Accepted:30 May 2021

Corresponding author:HALA, M. ISMAIL

E-mail address:ibrahimsabry.gravena@gmail.com

Present address:Pathology Unit, (Animal Health Research Institute, Mansoura Lab), Agriculture Research Center (ARC).

INTRODUCTION

Calf diarrhea is a multifactorial disease anda major problem in livestock production in Egypt and throughout the world (Ibrahim, 2007)which have serious financial and animal welfare implications inboth dairy and beef sucker herds(Uhdeet al.,2008). It has been detected that 75% of early calf mortality in dairy herds is caused byacute diarrhea in the pre-weaning period, alsostill amajor cause of economic loss to cattleproducers worldwide (Bartels et al., 2010).The aetiology of diarrhea is complex involving management, environmental, nutritional, physiological variations and variety of pathogens (Prescott et al., 2008), but80% of tested diarrheic calves indicated that infectious factor is still a major cause of calf diarrhea (Meir et al.,2010). The majority of diarrheic cases were identified among 0 to 4 week old calves (Wudu et al., 2008).Although E. coli and Salmonella are known to be the most common and economically important agents (Acháet al., 2004),Campylobacter spp, principally C.jejuniis among the main causes of gastroenteritis in newly born calves worldwide.Moreover, it is important human pathogens that may cause outbreaks of food-borne diseases and thus are of high public health importance(Cho,2012). Raw milk acts as the main source for Campylobacter spp. and primarily to be contaminated by bovine feces or direct contamination of milk as a consequence of mastitis (Modiet al., 2015).

Campylobacter isolation and identification is considered the standard method for disease identification; however, it is laborious due to the complex nature of Campylobacter(Li et al., 2014).Thus, molecular techniques, such as polymerase chain reaction (PCR) and sequencing, can permit the simple, fast and exact identification of C. jejuniand reveals its epidemiological characteristics (Miller et al., 2010).

The disease seriousness relies upon the virulence of the strain and on the host’s immune state(Youniset al., 2018).CadFis one of the reference virulence genes that encodes proteins involved in the attack and attachment of C. jejuni(Elmali& Can, 2019),and this gene is a highly prevalent in C. jejuniisolatesand have been proposed to play a role in enteritis and colonization in the luminal surface of the small bowel (Andrzejewskaet al., 2015).

Antimicrobial susceptibility test represents one of the most important an epidemiological tool and definition of the proper treatment of infections, consequently preventing or creating strategies that minimize the dissemination of resistant bacterial strains, mainly multiresistant ones especially Campylobacter spp(Shang et al., 2016),which has a potentially serious impact on food safty in both animal and human health (Hagoset al., 2021).

Despite extensive study, little is understood about the mechanism by which C. jejunicauses diarrheal disease, so various small animal models have been reported to study the process of enteroinvasiveness by C. jejuni(Heimesaatet al., 2014).The gastroenteritis due to Campylobacter ranges from mild to severe diarrheal disease (often bloody diarrhea), other symptoms are cramping, abdominal pain and fever within 2-5 days after exposure to the organism, which typically lasting 1 week with Complications (Murray et al., 2007),which attributed tovirulence factors that could play a role of colonization, adherence, and invasion of epithelial cells in the animal and human being(Wilson et al., 2010).

The aim of this study were to isolate and identify C. jejunifrom naturally infected calves and raw milk, and to determine the antibiotic susceptibility pattern for perfect control and treatment.Moreover, using an experimental rabbit model to study histopathological, immunohistochemichal and ultrastructural changes, as well as to enable testing of new therapeutics to prevent and/or combat infection.

MATERIAL AND METHODS

1.Samples collection

Atotal of 140 samples including raw milk (40) and sterile cotton rectal swabs(100) were collected from different farms exhibited severe diarrhea in newly born calves at Dakahlia Governorate in the period from December 2019 to April 2020. All samples were collected in sterilized bottles and were immediatelytransported to the laboratory in an insulated ice box at 4C within 1-2 h from collection and processed immediately upon arrival for isolation.

2.CampylobacterIsolation

Samples were examined for the presence of Campylobacter spp. using selective enrichment and isolation protocol recommended by Roberts and Greenwood (2003).One ml. of the homogenized samples was aseptically inoculated into sterile screw capped tube, containing 9ml of Bolton broth (Oxoid Ltd, Basingstoke, Hampshire, England) containing 5% laked horse blood and Bolton broth selective supplement which incubated under appropriate microaerophilic conditions in anaerobic jar by using the Gas Pack System BBL (5% O2, 10% CO2 and 85% N2) at 37˚C for about 4 hours prior to increasing the temperature to 41.5˚C for the remainder of the 48 hours of the incubation time for resuscitation. Loopful of the incubated broth was plated onto modified Charcoal CefoperazoneDeoxycholate Agar (mCCDA, Oxoid) with CCDA selective supplement and the plates were incubated for 48 hours at 41.5˚C under appropriate microaerophilic conditions, suspected colonies were selected and isolated.

3.Campylobacter Bacteriological Identification

Presumptive colonies of Campylobacter spp. were subjected to standard biochemical tests(Foster et al.,2004), including oxidase test, catalase production test, nitrate reduction test, hydrogen sulphide production using lead acetate paper, glycine tolerance test, NaCl 3.5% tolerance test, sensitivity to Nalidixic acid and Cephalothin and Hippurate hydrolysis test. Biochemically identified C. jejunicolonieswere stored at -70 °C in nutrient broths with 15% glycerol until subjected to molecular PCR identification.

4.Molecular characterization of C.jejuni

4.1.DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations.

4.2.Polymerase chain reaction (mapA gene)

The amplification of the mapAgene for C. jejuniwas carried out on 10 representative isolates that were biochemically confirmed utilizing the primers listed in Table 1. Amplification conditions were as follows: 6 minutes at 94 °C; 35 cycles of 50 seconds at 94 °C, 40 seconds at 57 °C, and 50 seconds at 72 °C; and a final extension of 3 minutes at 72 °C. The PCR products were analysed using 1.5% agarose gel electrophoresis ((Applichem, Germany, GmbH). Gelpilot 100 bp plus ladder (Qiagen, Gmbh, Germany) and Generuler 100 bp ladder (Fermentas, Thermo) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software(Shin & Lee, 2009).

4.3.Virulence gene characterization of C. jejuniisolates

The biochemically and molecular confirmed C. jejuniisolates were characterized for recognition of the cadFvirulence gene by PCR (Konkelet al., 1999) utilizing the primers listed in Table 1.

Table (1): Primers sequences, target genes, amplicon sizes and cycling conditions of C.jejuni.

5.Antimicrobial susceptibilitytest of C.jejuni isolate

All C.jejuni Confirmed isolates were screened for antimicrobial susceptibility using the stander agar disc diffusion technique as recommended by Clinical and Laboratory Standards Institutions(CLSI, 2014)for susceptibility to 8 different antibiotic disc: Amoxicillin(10μg), Ampicillin (10μg), Chloramphenicol (30μg), Erythromycin (15μg), Gentamycin (10μg), Norfloxacin (10μg), Sulfamethoxazole-trimethoprim (25μg), Streptomycin (10 μg). After 48h of microaerophilic incubation at 37˚C, the clear zones diameter for individualantimicrobial agents were measured and then translated into Sensitive (S) and Resistant (R) categories.

6. Experimental study

6.1. Experimental animal

Atotal of 60 infant rabbits at 10 days oldand C.jejuni pathogen free, were obtained from Rabbit production unit, Faculty of Agriculture, Mansoura University. To ensure the absence of Campylobacter infection, rectal swabs were performed immediately after reception of rabbits and plated on sheep blood agar plates containing cefoperazone, vancomycin, and amphotericin B (CVA agar). Rabbits were housed in cages with pads in the bottom. Food and water were provided at libitum, and were allowed to acclimate for 2 days before experiment.

6.2. Experimental design and infection

Rabbits were divided into 3 groups, each group has 20 rabbits. Group1:infected untreated withorogastric inoculationwith 1 × 10⁹ colony-forming units (cfu) of C. jejuni that were isolated previously and fully identified from naturally  infected calves and contaminated raw milk.Group 2:infected and treated with Norfloxacine within 12hr PI (1cm/kg). Group3:control group.

6.3.Clinical signs and postmortem:

Rabbits were observed daily for signs of diarrhea or death. Rectal swabs for bacterial re-isolation were obtained daily until the end of the 2-week observation period.Complete postmortemexaminations were done on infected and control animals.

6.4.Fecal inflammatory markers:Were measured according toNemelkaet al. (2009),a stool sample is collected daily in a clean container provided by the laboratory. This sample should be uncontaminated by urine or water.Occult blood (Hemoccult, Beckman Coulter Kit) designed to evaluate stool samples for hidden (occult) blood by measuring the heme (non-protein) part of hemoglobin from blood in the stool.Lactoferrin (Leuko-Test),is a glycoprotein present in activated neutrophils when the intestines are inflamed and are shed in stool. Gross blood was detected in stool by nacked eye.

6.5.Histopathological examination:

Three rabbits from each group were sacrificed each 12hr post infectionuntil five days, then once each 24hr till the end of experiment.The intestines were removed immediately. Specimens were placed in formalin 10%.Representative sections were taken from eachspecimen and stained with hematoxylin and eosin(Bancroft et al., 2013).

6.6.immunohistochemical staining for C. jejuni

Sections were permeabilized with 0.1% Triton X-100 for 15 min, treated with 3.3% H2O2 for 15 min, and washed. Samples were blocked for 30 min with 5% bovine serum albumin and incubated for 1 hwith an in-house mouse polyclonal antiserum against C. jejunior without serum as a control. Samples were thenincubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000; Sigma) for 1 h, developed with3-amino-9-ethylcarbazole,counterstained with hematoxylin, and mounted with aqueous mounting medium(Shang et al., 2016).

6.7.Ultra structural examination of intestine:

For electronmicroscopy, fragmentsof duodenum, ileum and colon were removedand fixed for 24 h at 4°C in callidine buffercontaining glutaraldehyde 2.5%. Samples were then washed in buffer, fixed for 1 h in osmiumtetroxide 1%, dehydrated, embedded in Epon (Merck), and cut with an ultramicrotome.Semi-thin sections were stained with toluidin blue; ultrathin sections were contrasted with uranylacetate and lead citrate, before examination with a Philips EM300 electronmicroscope (Electron Microscopy Unit, Mansoura University).

RESULTS

1.Campylobacter isolation and bacteriological identification

From the total of 140 collected samples, 41 campylobacter strains were isolated as following: 32 out of 100 (32%)calve fecal samples and 9 out of 40 (22.5%) raw milk samples from the same farms, by cultural methods.Campylobacter.spp. appear as grey color spreading colonies on blood agar media after incubation for 48hrs at 37c in microaerophilic condition (10% CO₂, 5% O₂ and 58% N₂). Gram staining examination showed pink color spiral rods were arranged as a single or in pairsunder microscope (100x). All isolated strains of Campylobacter spp were then subjected to biochemical tests, which were  positivehippurate hydrolysis, catalase test and indoxyl acetate hydrolysis.

2.Molecular Identification of Campylobacter jejuni isolates

Seven representative biochemically validated Campylobacter isolates were further molecularly identified through the amplification of map A gene specific to C.jejuni. All isolates recorded the specific product for C.jejuni(589 bp), as shown in fig.1.

Figure 1: Amplification of the mapAgene of C. jejuniisolates. Lane 1: DNA ladder (100 bp.), lane 2: positive control, lane 3: negative control; lanes 4-10: positive C. jejuniisolates showing specific bands at 589 bp.

The virulence characterization of molecularlyidentified C.jejuni isolates revealed that eight (15.22%) carried the virulence cadF gene among 46 C. jejuni isolates and produced the expected product (400), figure 2.

Figure 2: Agarose gel electrophoresis of CadFgene PCR products in C. jejuniisolates: Lane 1: DNA ladder (100 bp), lanes 2: positive control, Lane 3: negative control, Lanes 4 -10: positive C. jejuniCadFgeneshowing specific bands at 400 bp.

3.Antibiogram profile of isolated C.jejuni   

The antimicrobial susceptibility test of molecularly identified C.jejuni isolates (7)were screened against 8 antibiotics. All strains were susceptible to Norfloxacine, Erythromycin, Sulfamethoxazole-Trimethoprime, and Gentamycin. However, isolates showed resistance to Ampicillin, and Amoxicillin, Table2.

Table 2:Antimicrobial susceptibility pattern of C.ejuniisolates identified by disc diffusion method.

4.Experimental study:

4.1.Clinical signs after orogastric inoculation of C. jejuniinto rabbits:

After oral challenge with 10⁹CFU of C. jejuni, all 20 (100%) infected untreated rabbits(G1) were infected, and exhibited diarrhea after 12hr post infection which characterized by release ofloose, gelatinous unformed bloody stools followed by severe yellow diarrheal fluid.Duration of diarrhea varied between 1 to 4 days, and spontaneously remitted by day 6 after inoculation. None of the rabbits died but subsequently lost weight, off food and became very weak and lethargic. In contrast,in infected-treated group (Gr2),4of 20 (20%) of rabbits exhibited mild manifestation in the form of yellow watery diarrheaafter 12hr post infection but did not develope to severe illness andcompletlyrecovered after48hr.In control group (Gr3), appeared normal and did not exhibit any signs of illness, the change in their fecal consistency was not noted throughout the experiment.

4.2.Fecal inflammatory markers

Lactoferrin, gross blood and occult bloodwere measured in feces to investigate the degree of inflammatory response to C. jejuniinfection in each group as showed in table (3).

Table 3:Fecal inflammatoryresponse of C. jejuniexperimental infected rabbits.

All markers were negative on days 6 and 7 in the collected sample stool.

All infected untreated rabbits that developed diarrhea(100%) showed gross blood in their stool, this value gradually declined to35% by day 3, but no gross blood was present beyondday 5. Whereas the infected treated group revealed gross blood in (15%) of animals and disappeared in day 2 of experiment. In control group, all fecal inflammatory markers were negative during the study period.lactoferrin was more sensitive compared with gross and occult blood in detecting inflammation and antimicrobial susceptibility.

4.3.Gross examination:

The intestine of most infected untreated rabbits (G1)revealed acute enterocolitis indicated by bloody content,hyperemia, petechial hemorrhage and swollen. Small intestine, ceca and proximal colon were distended and fluid-filled (Fig3. A), compared with infected treatedrabbits (G2) which exhibit mild gross lesions andcontrol group that show normal intestine.

Figure(3):Gross findings in rabbits inoculated with C. jejuniisolated fromnaturally infected calves and cow raw milk. A. The infected untreated rabbits (G1):Showingcongested, swollen,distended and fluid-filled intestine. B. infected treated rabbits (G2):Showing mild focal congestion and not distended with fluids. C. Control group (G3):Showingnormal intestine.

4.4.Histopathology:

The abnormalities of histological analysis of infected rabbits were recorded mainly in the distal small intestine as well as in the colon. Lesions in infected untreated rabbits revealedcharacteristic progressive changes of the intestinal epithelial morphology. At 12 hr PI, intestinal villi appeared mostly normal with intact lining epithelium(Fig4.A). by24hr, heterophils were observed in the lamina propria of villi, withsmall clusters of bacteria were attachedand associated with erosions in theepithelial surface (Fig4.B).However most of the lining epithelial layer remained intact except little debris was observed in the lumen. Untillthat point there was no evident ofextensive disruption and disintegration of villus epithelium. By 36hr PI, we assessed gradual histopathological changes:the erosionswere more pronounced, causing cavities in the epithelialsurface and resulting in loss of the actin ring thatencircles and protect the villus, epithelial cells became extruding and unprotected which enhance epithelial hyperplasia, at that time luminal depris as well as heterophilsand bacteria became more abundant in the lumen and lamina propria(Fig4,C).But by 48hr till 72hr PI there was widespreaddamaged, degenerated, and sloughed intestinal mucosa,marked congestion of capillaries in the villi, severe epithelial hyperplasia leading to ulceration, serosal hemorrhage and diffuse polymorphonuclearneutrophilic infiltration (PMN) at the crypt epithelium.Loss goblet cell and crypt, smucosal ulceration, submucosal congestion and edema were extensive.Moreover,the inflammation extended from the submucosa to the muscle layer(Fig4.D).By the day 5 PI, the severity of intestinal lesions decreased and the damaged epithelial cells from the villus is balanced by proliferation in the crypts(Fig4.E) by day 7 PI, villus epithelial coverage was nearly normal and absence of any significant changes (Fig4.F).

The infected treated group(Fig 5), approximately 20% of rabbits developed clinical symptoms of C. jejuni. By 24hr PI, the intestinal villi and epithelial coverage were normal with normal mucosa, submucosa and muscularis(Fig5.A). By 36hr PI,exhibited rathermild histopathological changes suchas focal damage of epithelial lining villiassociated with small clusters of bacteria and heterophilic aggregation in the lamina properia(Fig5.B).By 48hr PI, congestion, edema in lamina properia,singleto mild scattered cell infiltrates inmucosa and lamina propria were recorded, but the lesions did not extend to submucosa and muscularis, normal goblet cells and crypts (Fig5.C).By day 3PIrecovery phase occurred and characterized by regeneration of villus epithelium with absence of congestion and edema (Fig5.D). By day 4PI, complete regeneration of lamina properia of villi with marked increase of villus length and width(Fig5.E). By day 5PI,intestineshowed conical shaped villi lining with simple columnar epithelium, with marked increase of villus width and length, normal cryptal glands and goblet cells were seen(Fig5.F).

The comparative histopathological evaluation between infected, infected treated and control groups were illustrated in table (4).

Table 4:Comparision of histopathological findings in all experimental groups:

Figure 4: Histopathological changes in the distal small intestine of rabbits infected with C.jejuni isolated from naturally infected calves and raw milk. A: by 12hr PI, intestinal surface appear normal and smooth with intact villi and lining epithelium, normal mucosa and submucosa (arrow),H&E, x20. B: by 24hr PI, erosion of epithelial surface associated with bacterial clusters (thin arrow) and mild heterophilic aggregation in the lamina propria (thick arrow),H&E, _x20. C: by 36hr PI, sloughed or desquamated villus epithelium associated with heterophilic aggregation(star), attached bacteria (arrow head),heterophilic aggregation in the lamina properia (thin arrow), H&E, _x40.D: by 48hr PI, degeneration and sloughing of villus epithelium (thin arrow). Congestion of blood vessels and infiltration with PMN in the lamina propria (star), submucosal congestion and edema(thick arrow), Congestion and edema in the muscularisexternae (arrow head), H&E,x40.E: by the day 5 PI, regeneration of the damaged villus epithelium (thin arrow), by proliferation in the crypts (thick arrow). F: by day 7 epithelial coverage appear nearly normal with marked increase in length and width of villi (arrow), H&E, x₁₀₀.

Figure 5: Histopathological changes in the distal small intestine of rabbits infected with C.jejuni isolated from naturally infected calves and raw milk and treated withNorfloxacine. A:by 12hr PI, normal intestinal villi with intact epithelial coverage (arrow) H&E, x10. B: by 24hr PI, focal damage of epithelial lining villi (thin arrow), associated with small clusters of bacteria and heterophilic aggregation in the lamina properia (thick arrow) H&E, x40. C: by 48hr PI, mild congestion, edema and single scattered cell infiltrates in lamina propria (thin arrow), normal submucosa and muscularis, normal goblet cells and crypts (thick arrow) H&E, x40.D:by day 3PI, regeneration of villus epitheliumand absence ofcongestion and edema (arrow) H&E, x40.E: by day4, normal lamina properia of villi with marked increase in length (arrow), H&E, x40.F: by day 5PI,showing conical shaped villi lining with simple columnar epithelium (thin arrow), with marked increase of villus width and length, normal cryptal glands and goblet cells (thick arrow) H&E, x40.

4.5.Immunohistochemistry:

The intestinal tissue sections from all groups were immunostained withC. jejuniantibody. By 24hr PI, In the infectedrabbits (G1& G2), C. jejuniantigen were detected through the lumen of the small and large intestines as well asbetween enterocytes, or intestinal absorptive cells, are simple columnar epithelial cells which line the inner surface of the small and large intestines(fig.6A).But,by 48hr PI, the infected untreated group (G1),immunoperoxidase labeling was intense in the mucosalregion and this intense labelingextended into thesubmucosa andmuscularisas well as the paracellular junction and at the basolateral surface of the epithelium.(Fig. 6B).However, by 48hr PIin the infected treated group (G2),immunostainedsectionsshowedimmunoperoxidase labeling in the mucosa only and not extended to ather layers of intestine(fig.6C).The tissue sections of negative control group showed no immunoperoxidase labeling wasseen(fig.6D).

Figure6:Immunohistopathological staining of rabbit intestinal tissue sections infected with C.jejuni isolated from naturally infected calves and raw milk: A:by 24hr PI, in the infected rabbits (G1&G2) showing presence of C.jejunithrough the luminal surface of intestine (thick arrow) as well as between enterocytes which lining the inner surface of villi (thin arrow).X40.B:by 48hr PI, in the infected untreated rabbits showing diffuse brownimmunoperoxidase reaction in the cell surface and mucosa (thin arrow) x40.The reaction extended to the submucosa and muscularis (thick arrow). C:by 48hr PI in the infected treated treated rabbits showed a significant immunoperoxidase reaction on the cell surface (thick arrow), and also labelingwithin the mucosa (thin arrow), but not extended to submucosa and muscularis.D:no positive brown staining reaction are seen in negative control rabbits.

3.4.6.Ultrastructural changes in intestinal epithelial cells induced by C.jejuniin rabbits:

Transmission electron microscopy (EM) was used to further study how C. jejuniinteracts with host epithelial cells of small intestine post infection. Transmission EM showed thatC. jejunidid not induce any structural changes in the epithelium during the first 12 h post infection. MoreoverC. jejuniwas not observed by EM in the lumen, tight junctions, desmosomes, or villus brush border, which remained intact in all experimental groups (Fig. 7A). By 24hr PI,numerous clusters of bacteria were observed in the lumen and closely attached to the villus tips causingpartial destruction of the brush borderwhile the adjacent epithelium without bacteria appeared normal (fig.7B).By 36hr PI, intraepithelial lymphocytic infiltration were stimulated, cellular swelling and edema were also observedwith cavities at the sight of attachment for invasion(Fig. 7C).By 48hr PI, the cellular membrane next to bacteria was invaginated, followed by diffuse disorganization of the epithelium architecture, and degeneration in the untreated group(fig.7D). By 60hr PI and after invasion, bacteria were surrounded by enterocyte vacules in which most of them lysed in the treated group. While in the untreated group retained their shape and size (fig.7E). By 72hr PI, the bacterial invasion fllowed by severe intracellular lymphocytic infiltrationand cell swelling in the untreated group(fig.7F).

Figure7: Transmission electron microscopy of the distal small intestine of rabbitsinoculated with C. jejuni. A:by 12hr PI, C. jejuniare not observed in lumen and brush border remain intact with tight junction between villi (arrow),X54,000. B: by 24hr PI,clusters bacteria attached to the epithelial cell surfaceand associated with loss of microvilli at the site of attachment (arrow),while the adjacent epithelium without bacteria appeared normal and intact (thick arrow),X35,000. C.By 36hr PI, C.jejuni closely attached to epithelial surface causing cavities for invasion(arrow), X 54,000 .D:by 48hr PI, C.jejuniaccumulated in the cavitiesof invasion below the normal level of adjacent intact epithelium (arrow), X 54,000.E:by60hr PI, intracellular C.jejuniapparently free in the cytoplasmwere surrounded by enterocyte vacules (arrow). mitochondria (star).X 35,000. F:by 72hr PI, bacterial invasion Followed bycell swelling, edema and severe inflammatory infiltrate of intraepithelial lymphocytes, (arrow), X65,000.

DISCUSSION

The main goal of this work was to determine the virulenceof C. jejuniand the post infection pathogenesis as well as antimicrobial resistanceas part of the characterization of this strain for future treatment planning and efficacy studies.

C.jejuni is generally considered commensals of livestock and did not normally cause clinical disease in adult animals, but now has emerged as one of the most common causes of serious foodbornzoonotic diseases worldwide for both human and animal and is responsible for gastroenteritis in young livestock in many industrial countries (Kaakoushet al., 2015).In the current study, Cultural examination, staining characteristics, biochemical tests and finally PCR were performed for the characterization of the Campylobacter spp. and the colony characteristics were exhibited grey color which was supported by Kabiret al.(2015) and Mehedulet al. (2018).The routine isolation and identification of Campylobacter spp. in laboratories were conducted on the basis of cultural and biochemical methods which was supported by Jamshidiet al. (2008).Hippurate hydrolysis test was used for discriminating between C. jejuniand C. coli which was also used by several researchers (Kabiret al., 2014 and Shiramaruet al., 2012).The current study recorded 32 (32%) and 9 (22.5%) Campylobacter spp. from 100 fecal and 40 raw milk samples respectively during the study period.The PCR is adefinitive, reliable, easy method and is required to facilitate rapid identification for C.jejuni(El-Kholyet al., 2016).In the current study, seven C.jejuniisolates that were biochemically identified were further molecularly characterized by mapA gene amplification specific to C.jejuni, all the isolates demonstrated positive specific product (589bp) for C.jejuniwhich is supported by Ghoneimet al. (2020). With respection to the virulence properties of C.jejuni, all the seven isolates carried virulence campylobacter adhesion fibronectin (the cadF) and generated the expected product (400bp), which agree with Elsayedet al. (2019)who reported that cadF is the most virulent gene campylobacter adhesion.

Antimicrobial susceptibility test represents one of the most important tasks of the clinical microbiology laboratory especially in veterinary medicine, because of the interrelation between resistance found in strains of animal origin and humans.Alsoit can be used as an epidemiological tool and for the definition of the proper treatment of infections,consequently preventing or creating strategies that minimize the dissemination of resistant bacterial strains, mainly multiresistant ones of C.jejuni(Hagoset al., 2021)which is emerged by WHO as aproblem of puplic health importance(Heredia and Garcia, 2018).

In the current study, 7C.jejuniisolates were investigated for their antimicrobial sensitivity pattern. The percentage of ampicillin and amoxicillin resistant C.jejuniisolates were 85% and 57%, respectively, and highest susceptibility percent with norfloxacine and erythromycin 85% and 71.4% respectively even with multidrug resistance isolates. This was in agreement with Faris(2015)who reported 97.2% and 83.3% for ampicillin and amoxicillin, respectively. Moreover, Macrolides and Fluoroquinolones are usually the drug of choice for treatment C.jejuni isolates.

In the current study, we developed a simple rabbits model of campylobacteriosis induced intestinal pathology and diarrhea.This experimental model enabled us to define several key features of the pathogenesis. Rabbits developed diarrhea within 48 h after oral inoculation of live C. jejunicontained mucus and gross blood. The clinical signs and pathologicallesions produced in the rabbits like those observed in humans and other large-animal models.C. jejunimultiplied in the distal small intestine and cecum of rabbits with largepopulations of C. jejuni, indicating its possible role in the pathogenesis of campylobacteriosis(Shang et al., 2016).Although diarrhea resolved spontaneously by day4post infection, the untreated rabbits may excrete bacteria asymptomatically for as long as 2 wk, these results supported by Black et al. (1988); Wassenaar and Blaser (1999).In the current study, the inflammatory nature of the diarrhea was further confirmed by the presence of lactoferrin and occult blood in stools. Compared with occult blood, lactoferrin was more sensitivein detecting inflammation that agreed with Stintzi (2005). Fecal inflammatory markers were highly positive in G1(infected untreated), whereas were mild in G2(treated), and negative in G3(control). These results agreed with Cook et al.(2020)who reported that fecal biomarkers result reflects the underlying GIT inflammatory conditions, moreover it can be used as amonitor response to intestinal therapy.

In the current study, infected untreated rabbits showed marked disruption of the villous epithelial surface in the small intestine, damage of the brush border was noted  in areas adjacent to bacteria that appeared to be bound to the epithelial cells, all appear to contribute to villus disruption and the breakdown of epithelial barrier function, this may be attributed to effacement of microvilli, re-distribution of cytoskeletal and tight junction proteins, and extrusion of epithelial cells in the small intestine as recorded by Nemelkaet al. (2009). Our histopathological finding revealed that microbial activities occurring mainly at the intestinal level and more significant than extraintestinal invasion as no histologic changes were seen in livers, kidneys, lungs, or mesenteric lymph nodes, that agreed with Shang et al. (2016)who reported that peritoneal histopathologic evaluation revealed no significant changes in infected animals at anytime during the study.

In the current study, based on immunohistochemical staining, we suggested that C.jejuni is capable of invading intestinal tissues. In the infected untreated rabbits, stained bacterial cells were observed in deep tissues as well as the paracellular junction of the epithelium and extended to muscularis compared to treated group which observed in lamina properia and mucosae only, similarly with Nemelkaet al. (2009)who reported the immunohistochemical stained C. jejuni(brown stain) can be seen in deep tissues of infected rabbit.

In the current study, for further understanding how C. jejuniinteracts with host epithelial cells, we visualized the epithelium in the small intestine of infected rabbits by electron microscopy (EM), which revealed that adherence would be the first step of invasiveness as C. jejunidid not induce any structural changes in the epithelium during the first 12 h after infection and C. jejuniwas not observed in the lumen, tight junctions which remained intact. However, clusters of attached bacteria were observed 24 h postinfection, particularly near the villus tips. Partial destruction of the brush border, infiltration of intraepithelial lymphocytes, and intercellular swelling were observed at 63hr PI. the epithelium was disorganized, and necrotic with severe infiltration of intraepithelial lymphocytes was observed at 72 hr PI, this results agree with Shang et al.(2016).

CONCLUSION

The implementation of hyagenic practices during milking and proper handling of milk during calves feeding, regular monitoring of antibiogram profile are very crucial in preventing C.jejuni infection, colonization and intestinal damage and subsequently economic loss in dairy farm.suggesting that experimental models may be useful to study the mechanisms and the pathogenesis of C. jejuni-induced intestinal disease.

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