HISTOPATHOLOGICAL CHANGES IN EXPERIMENTALY INFECTED MICE WITH PCR IDENTIFIED Corynembacterium psuedotuberculosis

This study was done to investigate the histopathological responses of the early phase during experimental Corynebacterium pseudotuberculosis infection in white mice. Corynebacterium pseudotuberculosis strains were isolated from natural cases of caseous lymphadenitis (CLA) in sheep. The experimental infection was done by using five isolates. These isolates were confirmed by PCR and they showed different biochemical characters. The experiment comprised five groups of five mice each; inoculated subcutaneously with C. pseudotuberculosis strains and observed for ten days following inoculation. There were different pathogical involvement of the Dalia A. Mohamed et al 16 isolates, these included; focal infiltration of polymorphoneuclear cells was seen in liver, lung, spleen, which indicated an acute infection process. Typical pyogranulomas with central necrosis and peripheral mononeuclear cells were observed in kidney clearly. These results illustrate the dual role of granulomatous lesions in chronic bacterial infections. Introduction Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CL); a disease characterized by the formation of suppurative abscesses particularly in superficial and internal lymph nodes and in internal organs in small ruminants. The disease occurs worldwide and causes significant economic losses particularly in sheep industry due to poor wool growth, decreased milk production, reproductive disorders, premature culling, carcass condemnation and rarely death (Alonso et al, 1992; Paton et al, 1994). The disease is considered to be one of the most economically important diseases of sheep and goats in Sudan, and emerged as a hurdle to the sheep export industry (Suleiman 2006). The objective of this study was to describe some aspects of pathogenicity and histopathology of PCR identified Corynebacterium pseudotuberculosis, during early phase of experimental infection in white mice. Materials and Methods Collection and Preparation of Samples: Three thousand and seventy five sheep carcasses were examined for the presence of abscesses in lymph nodes in Al Huda slaughterhouse at Omdurman Province Khartoum State. One hundred and fifty seven grossly enlarged lymph nodes were collected from one hundred and twenty nine infected sheep carcasses. The samples were labeled, stored on ice box and transported to the Bacteriology Department at Veterinary Research Institute, Khartoum, Sudan. U of K. J. Vet. Med. & Anim. prod. Vol. 2, No. 2, 2011 (15-24) 17 Culturing and Biochemical Identification: Pus samples from lymph node were inoculated on Blood Agar Base (Oxoid CM 271) supplemented with 7% defibrinated sheep blood. After the incubation of plates aerobically for 48 h at 37 0 C, small, white, dry and crumbly colonies were picked up for further identification. Pure cultures were prepared and identified according to Cowan and Steel (1974). Identification of Corynebacterium pseudotuberculosis Isolates by PCR: From forty nine isolates, five isolates were used for PCR. These were selected according to the differences in their biochemical characteristics. These isolates were 2, 11, 13, 64, and 65. Ten milliliter of fresh culture in Brain Heart Infusion broth (Biochemick 53286) were centrifuged at 4 0 C (2000xg for 20min) and chromosomal DNA extraction was performed by Quiageen kits. The primer used in this study was targeted on the 16S rRNA gene of Corynebacterium pseudotuberculosis and the sequence was selected from previously published work (Cetinkaya et al. 2002) and synthesized at Vivantis Biotech. Com. (Malaysia).The length of the primers was 20 mer and the annealing temperature was 55 o C. The PCR was performed in a thermocycler (PeQlab biotechnologie GmbH, Germany) in a final reaction volume of 25μl which contained 2.5 μl of PCR Buffer, MgCl 50mM (1 μl), 1 μl of each deoxynucleotide triphosphate, 0.4μl of Tag DNA polymerase, 1μl of each primer, 2.5μl of template DNA and 15.6μl distilled water. Amplification was obtained with 30 cycles following initial denaturating step at 94 o C for 5 min. Each cycle involved denaturation at 94 o C for 1 min., annealing at 56 o C for 1 min., and synthesis at 72 o C for 2 min. The amplified products were visualized by ethidium bromide staining after electrophoresis for 1h in 1.5% agarose gel. Experimental Infection in White Mice: This experiment was done using the PCR –confirmed five isolates. One colony of each strain from Blood Agar plates was inoculated in 5ml of sterile Brain Heart Infusion plus o.1% Tween 80 and incubated for 48 h at 37 o C. A dose of 0.2 ml (3,6 x 10 7 CFU) of fresh culture of each isolates were injected subcutaneously into five groups each of five white mice. Mice were obtained from Laboratory Animal Unit at Veterinary Research Institute. Dalia A. Mohamed et al 18 Control group was injected subcutaneously with 0.2 ml of sterile normal saline. Mice were observed for 10 days post inoculation for abscess development and death. Dead as well as surviving animals were necropsied and their internal organs (lung, heart, liver, spleen, and kidney) were inspected, smeared, Gram stained, and cultured on Blood Agar plates. Small parts of these organs were fixed in 10% formalin, dehydrated in a series of alcohol concentrations, embedded in paraffin wax, sectioned at thickness of 5 μm and stained with haemotoxylin and eosin (H&E). Results On microscopic examination Corynebacterium pseudotuberculosis was Gram-positive and small curved rod-shaped. Isolates which were positive for catalase, glucose, O.F., maltose and urease, inhibiting beta haemolysin of S. aureus and negative for oxidase, lactose, V.P., and nitrate were diagnosed as C. pseudotuberculosis. From one hundred and fifty seven abscesses examined in this study, Corynebacterium pseudotuberculosis was isolated from 49 samples (31.2%). Identification of Corynebacterium pseudotuberculosis by PCR: PCR products with the molecular size of 815 bp (Fig.1) were diagnosed as Corynebacterium pseudotuberculosis. The five strains were positive by PCR. U of K. J. Vet. Med. & Anim. prod. Vol. 2, No. 2, 2011 (15-24) 19 Fig.1.Identification of Corynebacterium pseudotuberculosis isolates by PCR Pathogenicity: Strains 2 and 13 were the most pathogenic as they caused acute disease and death in 2-5 days with 100% mortality rate. The organisms were demonstrated in Gram-stained smears and re-isolated from the livers, spleens, kidneys and lungs. Strains 11and 65 were less pathogenic, caused death in 47 days, had mortality rates of 80% and 60% respectively and the organisms were isolated from the internal organs on Blood agar plates. Strain 64 was not pathogenic but it was isolated from liver, lung, spleen and kidney. Post Mortem Findings: The internal organs in all white mice were congested and the spleen was enlarged and congested. M.W. +ve -ve 65 64 13 11 2