THE INTERACTION BETWEENTHE BOVINE CORONA VIRUS (BOCV) CAUSING RESPIRATORY INFECTION AND PASTEURELLA MULTOCIDA IN CALVES

THE INTERACTION BETWEENTHE BOVINE CORONA VIRUS (BOCV) CAUSING RESPIRATORY INFECTION AND PASTEURELLA MULTOCIDA IN CALVES

HALA, A. SALEM and A.M. NABIH

Animal Health research institute, Dokki Giza

Animal Reproduction Research institute, Harram Giza

Email: ashraf_nabih85@yahoo.com

INTRODUCTION

Bovine respiratory infections are frequently characterized by a primary viral infection followed by a secondary bacterial infection. One of viral pathogens implicated in this disease complex include bovine corona virus and one of most pathogenic bacterial agent implicated in acute and chronic BRD is Pasteurella multocida.

Bovine corona viruses (BCoV) cause respiratory and enteric infections in cattle and wild ruminants [Saif,  2007]. They belong to the Coronaviridae family in the Nidovirales order and are members of subgroup 2a along with swine hemagglutinating encephalomyelitis virus (HEV), canine respiratory CoV (CRCoV) and human CoV OC43 and HKU1. HEV, which causes wasting disease is an exception [Pensaert, 2006], the others cause enteric and/or respiratory disease. Recently discovered SARS-CoVs that are associated with both respiratory and enteric infections in humans and animals (civet cats, raccoon dogs, bats) belong to a new CoV subgroup 2b [,Saif,  2004-2007]. Unique to some group 2 CoVs including BCoV and wild ruminant CoVs, is the presence of a surface hemagglutinin-esterase (HE) glycoprotein (120–140 kDa). The HE acts as a receptor destroying enzyme (esterase) to reverse hemagglutination. Like other CoVs, BCoV possesses an outer surface spike (S) glycoprotein (190 kDa). Both elicit neutralizing antibodies that can block viral attachment and infectivity, so they are important for immunity and vaccines.

Pasteurella is a type of bacterial that commonly infects the respiratory tract of calves causing bovine respiratory disease. Pasteurella multocida is one of the most common bacteria isolated from calves suffering from shipping fever pneumonia. Pasteurella is usually a secondary bacterial invader, meaning that a virus or some other disease first weakens the immune system thus allowing Pasteurella to invade. Pasteurella is found throughout the environment and within the upper respiratory tract of cattle, but it usually does not cause disease in otherwise healthy animals. Thus, the concept has emerged that specific mechanisms must exist by which a primary BoCV infection can enhance bacterial colonization and virulence during a respiratory infection. Development of P.multocida-induced pneumonia is associated with environmental and stress factors such as shipping, and overcrowding as well as concurrent or predisposing viral or bacterial infections (Toply and Wilson 1998). The clinical presentation varied according to the age of the affected animal (Jacob et al., 2010). Pneumonic pasteurellosis is one of the most important disease complexes causing economic loss in the cattle feedlot industry. It is responsible for the largest cause of mortality in calves farms in Egypt. Infections with BoCV had not been considered in the past as an etiological factor in shipping fever pneumonia (SFP) of cattle Yates (1982). The objectives of the current investigations were aimed to examine nasal shedding of BoCV and P.multocida during the pathogenesis of acute, fatal BRD among 160 calves in 8 herds of calf-rearing units under research designed conditions, to quantitate the infectious loads of these viruses and bacteria in the mortality rate and to compare antibody responses to BoCV between fatal cases and clinically normal control calves befor and after medical treatment.

MATERIALS and METHODS

Animal and samples:

A total of  160  nasal swabs were collected from calves of ages ranged from 4 up 10 months old, from which 132 calves were suffered from acute respiratory manifestations, recumbency, anorexia, abdominal respiration as well as from their closely contact apparently normal calves (28 calves) from 8 herds at kaliobia, gharbia, sharquia   governorate and Miser-Alexandria road. The samples were collected during winter (january, up to April -2014). Two nasal swabs were collected aseptically from each examined calf, one sample in sterile bottles with PBS (PH 7.2) for virological examination, second swab was collected on nutrient broth for Pasteurella multocida cultivation.

Moreover one blood sample were collected from each examined calf for serum separation for serodiagnostic tests. Also two blood films were freshly prepared from each examined calf for diagnosis of Pasteurella multocida bipolarity. All samples were repeated 4 weeks after treatement with best choice antibiotics and avilable supportive treatements (analgesics and antipyretics), for bacteriological and virological investigations.

Bacterial isolation and identification:

Cultural and biochemical identification:

Nasal swabs which immersed in nutrient broth were cultured on brain heart infusion agar supplemented with 5% defebrinated sheep blood, blood agar and macconkey agar. Plates were incubated at 37˚C for 24 hours (Kodjo et al., 1999).

Pasteurella multocida were identified by colony morphology, Gram staining, and biochemical reactions,mostly includes oxidase, catalase, urease tests, triple sugar iron agar, motility tests, indol tests, voges proskauer and sugars fermintation tests (Atlas, 1997 and Baily and Scotts, 1998).

Blood films Staining:

The freshly prepared blood films from examined calves were stained with Leishman stain and examined under oil immersion lens for detection of Gram-ve biopolar bacilli.

Antimicrobial susceptibility test (Sensitivity test)

The susceptibilities of isolates to antimicrobial agents were determined by using the disk diffusion method according to the NCCLS (2002). The antimicrobial disk used are Ampicillin, Cefiquinome, Cephradine, Erythromycin, Ciprofloxacine, Amoxicillin / Clavulinic acid, Oxytetracyclin, Gentamycin, Pencillin G, Streptomycin, and Norfloxacine.

Viral isolation and identification:

1- Tissue culture

Madain Darby Bovine Kidney (MDBK) cell culture was obtained from virology department, Animal Health Research Institute, Dokki, Giza, Egypt.

2- Control sera

Positive and negative bovine sera against bovine corona viruse, was supplied by virology department Animal Health Research Institute, Dokki, Giza.

3- Virus Standard Mebus strain of bovine corona virus.The strain was obtained from Dr Linda Saif،s labaoratory in ohio Agricultural reseach and development Center, Wooster, USA were stored at -80 c in (AHRI) virology department.

4- Standard anti corona viruse conjugated with FITC used direct FAT supplied by central Vet. Lab. New. Haw. Webridge, UK.

5- Direct Fluorescent test (FAT) According to Payment and Trudel (1993), the test was carried out on fixed inoculated cell.

6- Polymerase Chain Reaction (RT PCR). The oligonucleotide primers used in the RT-PCR were designed from the published sequence of the N gene of the Mebus strain (Gen Bank accession No.M16620). The sequence of primers were as follows 5-GCAATCCAGTAGTAGAGCGT-3(21-40), and 5-CTTAGTGGCATCCTTGCCAA-3 (750-731). The predcted RT-PCR product size was 730 bp.

7- Virus neutralization (VN)

According to Storz, 2000

8- Infectivity neutralization assay. The IN titers in serum were expressed as the reciprocal of the serum dilution that completely inhibited cytopathic changes in 50% of the quadruplicates according to Storz, and Rott, 1981.

3. Bacteriological results:

Table 1: Pasteurella multocida isolates in diseased and apparently healthy calves befor treatement.

Table 2: Antimicrobial susceptibility tests of Pasteurella multocida.

Table 3: Number of isolted bovine respiratory corona viruses in diseased and apparently healthy calves befor treatement.

Table 4: Resultes of Infectivity neutralization (IN) testing to determine antibody titers of calves antisera against BoCV.

Table 5: Occurrence of mixed Pasteurella multocida and Bovine corona virus (BoCV) isolates in diseased and apparently healthy calves befor medical treatement with mortality rate.

Table 6: Occurrence of mixed Pasteurella multocida and Bovine corona virus (BoCV) isolates in diseased and apparently healthy calves after medical treatement with mortality rate.

Fig. (1): Infected MDBK cells showed intracytopalasmic bright fluorescent greenish granules (40X).

Fig. (2): PCRresult presenting the marker(M), positive control BCoV(1), resultes of two nasal swabs which appeared positive 730 bp(2-3) followed by negative control(4).

Bacteriologicl disscusion:-

Pasteurella multocida is a gram negative cocobacilli, non-motile, non-spore forming, facultative anaerobe from the family Pasteurellaceae. It is a normal inhabitant of the nasopharynx of healthy animals, but it is not a normal inhabitant of the bovine lung (Rice et al., 2008). Pasteurella multocida is a pathogenic bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, P.multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping (Debo et al., 2007). As shown in table (1), 43 Pasteurella multocida (32.60 %) were isolated from 132 diseased calves. While only 3 isolates were detected from 28 apparently healthy calves (10.70 %), under certain predisposing factors as shipping, rearing, transportation, overcrowding, mycoplasma infection and viral infection, Pasteurella multocida may shifting from being commensally to pathogen form (Confer  et al., 1995).

As shown in table (2), Antimicrobial susceptibility tests revealed that most of Pasteurella multocida were highly sensitive to Cefiquiome followed by Ciprofloxacin and Amoxicillin/ Clavulinic acid (86.70 %, 76.70% and 70.00%) respectively and highly resistant to Cephradin, Streptomycin, Oxytetracycline, Penicillin G. (83.30 %, 76.70 %, 70.00 % and 66.70 %) respectively. These results were nearly similar to that mentioned by Esaki et al. (2005), and catry et al. (2007) and disagree with Mevius and Hartman (2000) and Berge et al. (2006). The differences between our results and others may be attributed to many factors: misusing of antibiotics, individual physiological variation and differences in pathogenicity of the isolates and geographical localities.

After treatement of all infected groups with Cefiquinome antibiotic, (Cobactam 2.5 % - 1 Cm / 25 Kg body weight  once daily for 3 successive days) with  suitable, analgesic-antibyretic (twice a day), mortality rate were markadly decreased from 4 calves befor treatement (16 %) to only one calf (4.80 %) in case of Pasteurella multocida only  while in mixed infection mortality rate decreased from 6 calves (33.30 %) to one case (8.30 %) as shown in tables (5 & 6). These results were agreed with (Mevius and Hartman., 2000) who stated that, the immuno-suppression resulted from BoCV infection inhanced the susceptibility of the animal to infection with other microorganisms. They added that mixed infection of P.multocida and other microorganisms certainly lead to enhanced disease. Additionally, Shahriar et al. (2002) reported co-infection with BoCV and P.multocida in feedlot cattle with acute pneumonia. They concluded that the synergism between BoCV and other agents may complicate the disease condition. Which explain why the treatement with suitable antibiotic (Cefiquinome) together with suitable analgesic-antibyretic drug (Declo 5–1 Cm / 50 Kg body weight twice daily for 5 successive days) leads to enhansement of healthy condition of deseased calves and subsequently reduce the morbidity and  mortality rate as reported by Fatma    et al. (2008).

Interactions of virus and bacteria are important in developing respiratory manifestation and, making control of these diseases difficult. Strategies must control and prevent the primary agents (most commonly viral by vaccination) rather than simply treating the secondary agents that cause clinical diseas. Many bacterial infections are difficult to initiate without the presence of others stress factors specially viral infection mohamed et al. (2006).

Virological results and discussion:-

Bovine coronavirus (BCV), is associated with respiratory tract infections in calves and feedlot cattle. Cattle shedding BoCV nasally after entering the feedlot were at increased risk for respiratory disease Lathrop, (2000) and had high mortality to BCV infection Storz, (2000). Bovine corona virus is widespread in the cattle calves population, resulting in economic losses to the beef and dairy industry throughout the world Melanie and Sanjay (2010). Virus multiplication and shedding is highest during the early phase of infection when infected calves still sufering from symptoms Storz, (1998). 29 isolates of corona virus were detected befor treatement, and not isolated after one month post treatement. This agree with Mustafa et al. (2002) who found that the peak of the shedding time was at 4 days post starting of feed-lot and not shedding after 21 days. This agree with our result in table (3 & 6) befor and after medical treatement.

In our study, BoCV were isolated by inoculation on tissue culture (MDBK) and identify by VN, FA and PCR. Calves with respiratory symptom were examined for BoCV using MDBK, cell culture is the most sensitive method available to detect BoCV in naturally infected calves and many cytopathic viruses were successfully isolated from nasal swabs as reported by Manuel et al. (1999) CPE were characterzed by enlarged, round, detached dark cells were bserved at approximately 72 hours post inoculation of MDBK cell following 3 blind passages. The direct FAT method in the detection of BoCV is recommended for practical examination because of its simplicity as showed by Tsunemitsu    et al.  (1991). Detection of BoCV in nasal samples by direct FAT: BoCV antigens were detected after inoculation of nasal swabs on tissue culture. 27 of 132 (20.50%) (table 3)  shows specific fluorscene observed in MDBK cell culture stained with FITC - conjugated anti-BoCV antibody (Mebus strain) as shown in Fig.(1), the results of direct FAT of nasal swabs were in close agreement with the results of  the virus neutralization of positive samples which agree with Tsunemitsu et al. (1991).

We used RT-PCR for detection of virus for its high degree of sensitivity especially for specimens from those calves early or late in the course of illness or after reinfection which may have a low level of BoCV shedding. The number of nasal samples that positive by RT-PCR (29) was higher than those positive by VN and FAT (27) that suggests that RT-PCR is more sensitive as in table (3), and Fig.(2) same as identified by Cho et al. ( 2000 ). Normal calves had not developed signs of respiratory tract disease and not shed. The BoCV in nasal secretion so we could not isolate the BoCV table (3). This disagree with Robert et al. (2011) who isolated BoCV from both healthy and sick calves, and this agree with Storz et al. (2000) who not isolated BoCV from clinically normal cattle had significantly higher of Infectivity neutralization (IN) levels than the cattle developing clinical signs, suggesting that a high level of IN antibody against RBCV enabled the cattle to resist RBCV infections more efficiently, thus preventing clinical signs of respiratory tract disease as shown in table (4). The RBCV isolated from nasal swab expressed receptor-destroying enzyme (RDE) activities Storz (1996), Storz (1992) RDE functions mediated by an acetylesterase (AE), AE that hydrolyzes an ester bond to liberate acetate from sialic acid-containing bovine submaxillary mucin, a substance with a chemical composition resembling the glycocalyx that covers the bovine respiratory tracts Herrler, (1985), Storz, (1992). Pathogenetic mechanisms probably involved action of this viral enzyme by inducing glycocalyx changes that lowered mucosal resistance barriers and favored virus penetration and adhesion of P. multocida to cells of the lower respiratory tract. Because BCoV antibodies are widespread in cattle, paired acute and convalescent serum samples are needed for serologic diagnosis of BCoV infections. Detection of active infection by a 4-fold or higher rise in BoCV-neutralizing antibody titers in acute to convalescent samples (table 4). Diseased calves that were shedding BoCV at early stage of infection had BoCV antibody levels of 4-8, whereas healthy calves did not shed virus with BoCV antibody titers of 16 - 32 befor treatment. Significant increase in the level of antibody in serum was observed for all these calves after one month that agree with Xiaoqing et al. (2001). The antibody titers of diseased calves ranged from 32-256 and the 28 normal control calves that remained clinically healthy and did not nasally shed BoCV ranged from 16 to 128 after one month      (table 4).

Morbidity and mortality will be increased when a combined infection of virus and bacteria is present compared to an infection with either agent alone as shwon in table (5 & 6).

Implications- So current programs to prevent BoCV involve vaccination of pregnant cows and passive colostral protection of new borns. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature (Debo et al., 2007), for P. multocida control vaccination should be done 3 wks. before transport to the feedlot and can be repeated on arrival. In dairy calves, vaccination of the dam may be of benefit by providing passive immunity to the calf. H somni bacterins are available, and there is some evidence that they are effective in control of BRD in feedlot calves even when only 1 dose is given on arrival (Merck vet. Manual 2012).

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