Clinico-pathological Changes Associated with Brucella melitensis Infection and its Bacterial Lipopolysaccharides ( LPS ) in Male Mice

Brucella melitensis (B. melitensis) is gram negative, aerobic bacteria that cause Brucellosis in humans’ sheep and goats. Brucellosis causes abortion in wild and domestic animals resulting in enormous financial losses. Therefore, the purpose of this study was to evaluate the clinico-pathological changes associated with Brucella melitensis infection and its bacterial Lipopolysaccharides (LPS) in male mice. Three groups of 24 Balb/c male mice consisting of 8 mice in each group were used as an animal model for the study. The control group were inoculated intraperitoneally with 1 mL of Phosphate Buffered Solution (PBS) pH 7 while, the treatment groups were inoculated intraperitoneally with 1 mL×10 of B. melitensis colony and 1 mL×10 of Lipopolysaccharides (LPS) extracted from B. melitensis respectively. Mice that showed severe clinical signs and those that survived were euthanized by cervical dislocation method after 5 days of post infection subsequently, post mortem was conducted and histopathological studies were carried out. B. melitensis group showed severe clinical signs between 6 to 17 h of post inoculation compared to the PBS and LPS groups. The LPS group became lethargic 2 h post inoculation but, they become active after 5 h post inoculation, while the control group (PBS) exhibited normal responses. Histopathology results showed severe tissue alterations in the reproductive organs of the B. melitensis group compared to LPS group. In conclusion, the atrophy of the spermatocytes in the testes and degenerative necrosis of the pseudo stratified epithelium of the vas deferens in the B. melitensis group were severe while, LPS group showed moderate atrophy of the spermatocyte of the testes and severe degenerative necrosis of the pseudo stratified epithelium of the vas deferens.


INTRODUCTION
Brucella melitensis (B.melitensis) is gram negative, facultative intracellular coccobacillus or short rod (0.6-1.5 µm) aerobic bacteria that cause Brucellosis in humans, sheep and goats (Gupta et al., 2006;Joicy et al., 2012).The mucosal surface of the alimentary tract is the principal route of entry for B. melitensis (Carlos et al., 2011).Unlike many other pathogens, Brucella spp.invade host cells without activating innate immune defence systems and then resist intracellular killing to persist in the host (Gorvel and Moreno, 2002;Barquero et al., 2007).Brucellosis causes abortion in wild and domestic animals resulting in enormous financial losses especially in developing parts of the world (Andrew et al., 2003).
The most current threat is on the possible use of Brucella species, principally B. melitensis, as an agent of biological warfare because of the incapacitating ailment it causes.Extensive spreading of aerosolized B. melitensis would pose a biological, agricultural, as well as an economical threat to all countries engrossed (Gul and Khan, 2007).The disease is an emerging and re-emerging disease (Seleem et al., 2010).The morbidity of the disease reported to be increase in number in central Asia and certain countries in the Middle East (Pappas et al., 2006).
Brucella Lipopolysaccharides (LPS) is less endotoxic compared to enteric gram-negative bacteria, due to the presence of the unique components O-Polysaccharides (OPS) and Lipid A (Jarvis et al., 2002;Cardosa et al., 2006).The Brucella cell wall consists of a peptidoglycan layer which is strongly associated with the outer membrane.The outer membrane contains lipopolysaccharides, proteins and phospholipids (Galdiero et al., 1995). B. melitensis, B. abortus, B. Suis and B. Neotomae have smooth structure of LPS.The specific O-chain saccharides of the LPS structure determine the M (melitensis) type, or A (abortus) type antigenicity.Brucella antigenic characteristic of Brucella melitensis depends on LPS (Joicy et al., 2012).There is limited information of host cell response towards immunogen of B. melitensis in mice.Therefore, the objectives of the present study are to compare the clinical signs and cellular changes of mice with reference to male reproductive organs following inoculation with B. melitensis and its bacterial Lipopolysaccharide (LPS).

MATERIALS AND METHODS
Bacteria: Stock of B. melitensis was obtained from the Histopathology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM).The organism was re-cultured onto media in which biotin, thiamine and nicotimide were used as a supplement for bacterial growth.The incubation period of this bacterium is 3-4 days and the optimum temperature for the growth is 36-38 o C. All procedures and experiments illustrated were undertaken under a project license approved by Animal Utilization Protocol Committee with reference number: UPM/FPV/PS/3.2.1.551/AUP-R120.
Preparation of 1 mLx10 9 colony of Brucella melitensis: Mac Farland technique was used to determine the concentration of B. Melitensis (10 9 colonies).

LPS extraction from 10 9 of colony Brucella melitensis:
The LPS extraction kit (Intron biotechnology®) was used to prepare the LPS from B. melitensis bacteria.In the present study, 10 9 cfu of organism (B.melitensis) was extracted.The bacteria were harvested by centrifugation in room temperature at 13,000 rpm.Then, 1 mL lysis buffer was added and it was vortex vigorously.Thereafter, an amount of 200 μL of chloroform was added and it was vortexed vigorously for 10-20 sec after which it was incubated at room temperature for 5 min.Following this, it was centrifuged again at 13,000 rpm for 10 min at 4 o C. The supernatant of 400 μL was transferred to a 1.5 mL tube.800 μL Purification buffer was added into the tube and it was mixed well.Subsequently, it was incubated in -20 o C for 10 min.Then it was centrifuge at 13,000 rpm for 15 min at 4 o C. The upper layer was removed to obtain the LPS pellet.One millilitre of 70% ethanol was added to the pellet to wash the pellet.The 30 μL of 10 M Tris-HCL buffer pH 8 was added to LPS and was dissolved by boiling it for 2 min.
Experimental design: Balb/c mice were obtained from the Institute of Cancer Research (ICR).Twenty four Balb/c mice were divided into 3 groups, with 8 mice in each group.Mice in group 1 were inoculated intraperitoneally with 1.0 mL×10 9 of Phosphate Buffer Solution (PBS), pH 7, mice in group 2 were inoculated with 1.0 mLx10 9 of B. melitensis and group 3 were similarly inoculated with 1.0 mL×10 9 of Lipopolysaccharides (LPS) of B. melitensis colony.All the groups were observed for 120 h (5 days) for clinical signs such as ruffled hair, movement, ocular discharge, closed eyed and responsiveness.Mice showed severe clinical signs and those that survived were euthanized after 5 days via cervical dislocation method and the reproductive organs namely testes, seminal vesicle and vas deferens were obtained for histopathological study.

Clinical scoring:
The clinical signs of all the three groups of mice namely, control, whole bacterium (B.melitensis) and LPS were scored in scale of 0-3 based on the presence of ruffled fur, ability to move and discharges from the eye as well as the degree of responsiveness following infection of B. melitensis and its LPS.The score 0 represented no abnormality of clinical signs observed, 1 for mild, 2 for moderate and 3 for severe.
Lesion scoring: Cellular changes were scored following the evaluation of 10 slides.3 organs were examined.Lesion scoring was divided into 4 namely; 0 = normal; 1 = mild lesion less than 1/3 of the field was involved; 2 = moderate lesion between 1/3 and 2/3 of the field were involved; 3 = severe more than 2/3 of the field was involved.
Statistical analysis: Data obtained were analyzed using the statistical software package JMP 9 (SAS, QSAS Institute Inc, Cary, NC, USA).Analyses were considered as significant at p<0.05.

RESULTS
Clinical observation: All challenged groups showed significant clinical changes following infection with B. melitensis and its LPS.In Brucella group, the clinical signs did appear earlier and were severe in comparison to the control and LPS inoculated group.Lethargic and laboured breathing signs were exclusively noted at 11 h post-inoculation.The severity of the clinical sign reached its peak at 15 h when animals were humanly euthanized at this point.Brucella group showed significant changes in all clinical observations compared to the control and LPS group (Table 1).In contrast, animals treated with LPS extracted from B. melitensis exhibited the clinical signs at 12 h postinoculation.The severity of the clinical signs in this group ranged from mild to moderate with significant differences (Table 1).mice in the present study after 5 days of post inoculation with immunogen while, Fig. 1 to 6 showed the histopathological changes in the male reproductive organs of mice after 5 days of post inoculation with immunogen.There were significant differences in the

DISCUSSION
B. melitensis is a gram negative, coccobacillus or short rod bacteria (OIE, 2009;Joicy et al., 2012) which cause brucellosis in humans, sheep and goats.This bacterium is usually transmitted via contact with the placenta, fetus, fetal fluid and vaginal discharge.Goats are shedding this bacteria in vaginal discharge for about 2-3 months, while sheep for about 3 weeks.Sheep and goats were infected with these bacteria through the mucous membrane of oropharynx, upper respiratory tract and conjunctiva.B. Melitensis also can be transmitted through the broken skin.Male goats infected with these bacteria, resulted in the development of orchitis and epididymitis leading to infertility (Lilenbaum et al., 2007).Arthritis was also observed but occasionally.Goats were the source of the infection for human population through the consumption of goats' products (Wallach et al., 1997;De Massis et al., 2005;Kahler, 2000;Acha and Szyfres, 2003;Sriranganathan et al., 2009).
In the present study, mice inoculated with B. melitensis exhibited severe clinical signs and all the mice died gradually after 6 h and up to 15 h of post inoculation.In contrast, mice that were inoculated with LPS became lethargic 2 to 3 h post inoculation, but after that, they exhibited normal clinical signs.Similarly, in a study conducted by Alton (1990) in goats the infection lasted for a duration of short period of slight infection to persistence for years while sheep were resistance to the infection probably due to breed disposition.LPS group become more active at 12 h of post inoculation and all mice survived until the day they were euthanized.A study conducted by Edmond et al. (2002) proved that Brucella species lacking major outer membrane protein Omp 25 were attenuated in mice and protected them against B. melitensis.Furthermore, a study by Mina et al. (2004) stated that during infections by gram-negative bacteria, the presence of Lipopolysaccharide (LPS) stimulated the innate immune system whereby the inflammatory response played a critical role in helping to clear the bacteria and prevent infection.The mice in PBS group showed normal clinical signs throughout the 5 days of the experiment.
In the current study, mice in groups and LPS group showed atrophy of spermatocytes in the testes and also degenerative necrosis in the pseudo stratified epithelium of the vas deferens.Atrophy of the spermatocyte was also seen to increase the space between the spermatocytes as shown in (Fig. 1 and 2).In the degenerative necrotic cells there were ballooning appearances in the cytoplasm of the pseudo stratified epithelium cells as indicated in (Fig. 5 and 6).However, there were no lesions in the seminal vesicles as observed in the B. Melitensis group, LPS and PBS.In a previous study conducted by Izadjoo et al. (2008) stated that mice inoculated with B. melitensis (5 mL×10 11 ) via oral route, showed inflammation of testes on day 57, but, recovered on day 88.In the present study, inoculation with 1 mL×10 9 of B. melitensis intraperitoneally indicated the development of clinical signs and histopathologic lesion similar to the findings of Izadjoo et al. (2008).Additionally, Study by Takele et al. (2009) indicated that mice inoculated with 5×10 8 cfu B. melitensis colony showed clinical signs at the early stages such as extreme shivering, erection of hair coat, anorexia and dullness.These clinical signs virtually disappeared at the later stages after one month of post inoculation.
In another study conducted by Jinkyung et al. (2002) stated that mice inoculated with 5×10 5 of B. abortus intraperitoneally were unable to resolve the infection and died between 10-20 days post inoculation.In the present study, the mice were inoculated with 1 mL×10 9 B. melitensis intraperitoneally and they died within 15 h of post inoculation.
Another study conducted by Apurba et al. (2002) stated that the intranasal immunization of B. melitensis LPS with Neisseria meningitides Group B Outer Membrane Protein (GOMP) significantly protected the mice against the virulent.B. melitensis (strain 16 M).It reduced the dissemination of spleen and liver injury but not lung infection.LPS is indispensable for the viability of most Gram-negative bacteria.Brucella LPS is less endotoxic than that of enteric Gram-negative bacteria, a feature attributed to unique components in the OPS and lipid A (Jianwu and Thomas, 2011).
In conclusion, this experiment prove that mice infected intraperitoneally with B. melitensis developed severe clinical signs whereas mice inoculated with B. melitensis LPS developed normal to mild clinical signs.Furthermore, both B. melitensis immunogen inoculated in male mice developed pathological changes in the testes and vas deferens.The B. melitensis LPS could be a promising candididate for the development of vaccine against B. Melitensis in mice, sheep, goats and humans.

Fig. 4 :
Fig. 4: Normal photomicrograph of a section of seminal vesicle of mouse inoculated with LPS extracted from B. Melitensis (HE, bar = 50 µm) Table 2 showed the mean score of cellular changes of the reproductive organs of male

Table 1 :
Summary of clinical signs observed in mice inoculated with PBS, Brucella melitensis and LPS

Table 2 :
Mean score of cellular changes of reproductive organs observed in mice 5 days post inoculation with immunogen *: Significant value p<0.05;Comparison between immunogens group and control group