New sites of localisation of Pasteurella multocida B:2 in buffalo surviving experimental haemorrhagic septicaemia

Clinical responses

All calves of group 1 were euthanised following development of advanced clinical signs; two were euthanised at 24 h post-infection, while the rest were euthanised at 48 h post-infection. Affected calves appeared dull and depressed, recumbent, pyrexic, anorexic and dyspnoeic, with nasal discharge and diarrhoea.

On days 5, 6 and 7 after being mixed with the calves from group 1, three calves (2A, 2B and 2C) of group 2 were transiently pyretic, with average body temperatures of 40.4°C (SEM = 0.15), 40.6°C (SEM = 0.32) and 40.3°C (SEM = 0.50), respectively (Figure 1). Another calf (2D) showed progressive pyrexia for more than 48 h and was euthanised because of severe clinical signs. The surviving calves in group 2 eventually became carriers. None of the calves in group 3 showed clinical signs.

Gross pathology

In general, all calves of group 1 and one calf (2D) from group 2 that were euthanised following the development of severe clinical signs had classic lesions of acute HS, including severe congestion and haemorrhage of mucosal surfaces, subcutaneous tissues and respiratory, gastrointestinal and urinary tracts. Fibrin was deposited on the serosal surfaces of all thoracic and abdominal organs.

The surviving calves of group 2 had multifocal pulmonary congestion and pneumonia. Fibrin was deposited on the omentum, mesentery, peritoneum and serosal surfaces of the gastrointestinal tract. However, the mucosal surfaces of the gastrointestinal tract appeared normal apart from the rectum, which had mild multifocal haemorrhages.

Isolation and identification of P. multocidaB:2

Pasteurella multocida B:2 was successfully isolated from organ samples of all calves that were euthanised because of acute HS.

Immunoperoxidase examination

The respiratory, gastrointestinal and urinary tracts of all surviving group 2 calves had mild to moderate P. multocida B:2 immunoreactivity. Within the respiratory tract, immunoreactivity was noted in the nasal mucosa, tonsil, epiglottis, trachea and the lungs. In the nasal mucosa, the cells of nasal glands showed intracytoplasmic immunoreactivity (Figure 2A) while the tonsillar epithelium and tonsillar crypts had immunoreactivity amidst cellular debris (Figure 2B). Rare immunopositive bipolar rods were detected on the tonsillar epithelium and crypts.

Similar immunoreactivity was detected in the cells of serous glands, the epithelial cells of the epiglottis and on the epithelium of the trachea (Figure 2C). In the lungs, positive immunoreaction was observed on the surface of pneumocytes. Intracytoplasmic immunoreaction was observed in occasional infiltrating and degenerating alveolar macrophages (Figure 2D). Positive immunoreactivity was also detected in bronchiolar exudates that were mixed with desquamated bronchiolar epithelial cells.

Within the gastrointestinal tract, all organs had immunoreaction except for liver and mesenteric lymph nodes. The rumen, reticulum and omasum had positive immunoreactivity beneath the keratin layer (Figure 3A), while the abomasum was immunoreactive at the tips of the mucosal surface (Figure 3B). Similar positive reactions were found at the tips of the mucosal surfaces in all sections of small and large intestines and within rare infiltrating macrophages.

There was no immunoreactivity in any kidney samples. However, the ureter showed mild intracytoplasmic immunoreaction within the epithelial cells. Few immunopositive bipolar rods were detected in ureteral lumina (Figure 4A). Similar mild immunoreactions were observed in sections from the urinary bladder, particularly in the urothelial cells (Figure 4B).

There was no immunoreactivity in any tissues from calves in group 3 (Figure 5).

Animals

Twelve eight-month-old, clinically healthy, buffalo calves from a farm with no history of HS or vaccination against P. multocida were selected for the study. Calves were acclimatised for a period of 5 days and treated with anthelmintics. Nasal swabs were collected daily to ensure calves were free from P. multocida by culture and PCR [19]. Calves were fed cut grass supplemented with 400 g/calf/day of palm kernel-based pellets. Drinking water was available ad libitum. Access to veterinary care was available at all times and the well-being of the calves was assessed regularly.

Inoculum preparation

Stock cultures of P. multocida B:2 isolated from a previous outbreak of HS were used to prepare the inoculum. Bacteria were cultured onto blood agar plates and incubated at 37°C for 24 h. Four colonies were inoculated into brain-heart infusion broth and incubated at 37°C with shaking at 150 revolution per minute (rpm) for 18 h. Bacteria were quantified via serial dilution, and an infective inoculum containing 1.0 × 10⁵ colony forming units (cfu)/mL was prepared [20].

Experimental design

All experiments were approved by the Animal Care and Use Committee of Universiti Putra Malaysia (approval number 12R148).

Calves were divided into three equal groups. Group 1 served as the infected group, group 2 (consisting of calves 2A, 2B, 2C and 2D) as the commingling group and group 3 as the negative control group. All calves of group 1 were challenged subcutaneously at the shoulder with 5 mL of inoculum containing 1.0 × 10⁵ cfu/mL of P. multocida B:2 [21]. Calves of group 2 were not infected but were kept together and were allowed to commingle with the infected calves of group 1 [22]. Calves of group 3 were inoculated subcutaneously at the shoulder region with sterile phosphate-buffered saline and housed separately. Following inoculation, all calves were observed for clinical signs of HS and rectal temperatures were checked daily. Calves with advanced clinical signs were euthanised according to the guideline of the Ethics Committee, Universiti Putra Malaysia.

Deep nasal, rectal and vaginal swabs were collected daily from all animals, throughout the 43-day study period using sterile cotton swabs. Deep nasal swabs were obtained by cleaning the external nares with 70% alcohol, inserting the swabs 15 cm into the nostrils, and swabbing the nasal and nasopharyngeal mucosa by rotating the swabs several times. The rectal and vaginal swabs were obtained by cleaning the rectal and vaginal regions with 70% alcohol, inserting the swabs 7 cm into the rectum or vagina, and swabbing the vaginal and rectal mucosa by rotating the swabs several times. All swab samples were immediately transported to the laboratory in Cary Blaire medium.

Once a week had passed with no positive cultures, calves were injected intramuscularly with dexamethasone (Dexason; Troy Laboratories) for 3 consecutive days at the rate of 1 mg/kg to induce immunosuppression [12]. After the first set of injections, two additional series of dexamethasone injections followed at 7-day intervals.

After the final dexamethasone injection, all surviving calves in groups 2 and 3 were euthanised. Samples of nasal passage, tonsil, epiglottis, trachea, all eight lung lobes, liver, rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, mesenteric lymph nodes, caecum, colon, rectum, kidney, ureter and urinary bladder were collected from all euthanised animals in 10% neutral buffered formalin.

Tissue samples from group 1 were subjected to culture and identification of P. multocida B:2. Tissue samples from the surviving buffalo of group 2 and negative control of group 3 were subjected to culture and identification of P. multocida B:2, histopathologic and immunohistochemical examination.

Isolation and identification of P. multocidaB:2

All nasal, rectal and vaginal swabs were cultured on blood agar plates incubated at 37°C for 18 h. Identification of P. multocida B:2 was based on morphological criteria [23] and PCR [19]. Tissue samples were homogenised and diluted before deoxyribonucleic acid (DNA) extraction (NucleoSpin® Tissue; Macherey-Nagel, Düren, Germany). Purified DNA was subjected to detection by PCR [19].

Localisation of P. multocidaB:2

Tissue samples were fixed in 10% buffered formalin for at least 24 h before being trimmed, embedded in paraffin and sectioned at 3 μm. Sections were then subjected to immunoperoxidase and methylene blue stainings. Sections were dewaxed by incubation at 60°C for 15 min and immersion into xylene. Rehydration was via successive immersion in absolute alcohol, 90% alcohol, 70% alcohol and 50% alcohol. Sections were then exposed to citrate buffer using a carousel microwave at 50 W for 15 min and then Dako REAL™ Peroxidase Blocking Solution at room temperature for 30 min. Samples were incubated with primary rabbit hyperimmuneserum (1:200) against P. multocida B:2 at 37°C for 1 h, and then incubated with 1:1000 diluted peroxidase conjugated antiserum (polyclonal goat anti-rabbit immunoglobulin G (IgG); Dako) at 37°C for 30 min. Finally, sections were incubated with 3,3′-diaminbenzidine (DAB) (Dako Liquid DAB + SubstrateChromogen System) at room temperature for 2 min, followed by washing in distilled water and counterstaining with methylene blue. Finally, all sections were examined under a light microscope.

Adjacent sections were dewaxed and stained with methylene blue for 30 s before being examined under light microscope to detect bipolar P. multocida B:2.

Statistical analysis

The Student’s t test was used to compare rectal temperatures between the carriers and control calves.