Bacillus cereus nositelj plazmida pXO1 s genom pag uzrokuje u goveda s oslabljenim imunosnim sustavom smrtonosnu septikemiju sličnu bedrenici - kratko priopćenje.

Bacillus cereus is ubiquitous in nature and while most isolates appear to be harmless, some are associated with food-borne illnesses, wound infections, endocarditis, osteomyelitis, endophthalmitis and urinary tract infections in humans. Recently, a few isolates have been identified as the causative agents of anthrax-like severe pneumonia in humans, and these isolates were found to harbor most of the B. anthracis virulence plasmid pXO1. Here we report the characterization of three clinical B. cereus isolates recovered from heart blood and spleen samples of cattle which had died with ‘anthrax like’ symptoms. Apart from the cultural characterizations, primers targeting the 16S rRNA gene of B. cereus were designed and used on these isolates. The isolates were found to harbor the pXO1 plasmid and lacked pXO2 plasmid. Further characterization of the pXO1 plasmid revealed that the isolates contained pag, lef and cya genes, which code for protective antigen, lethal factor and edema factor toxins responsible for eliciting an ‘anthrax like disease’ in cattle. The sequencing and phylogenetic analysis of partial pag gene sequences of B. cereus isolates were identical to pag gene sequences on the pXO1 of B. anthracis. In a pathogenicity test on mice, B. cereus isolates, when inoculated by the intra peritoneal route, caused mortality of the mice within 6 hours post inoculation.Bacillus cereus je posvudašnja bakterija. Većina izolata te bakterije je neškodljiva, a neki mogu uzrokovati bolesti koje se prenose hranom, infekcije rana, endokarditis, osteomijelitis, endoftalmitis i infekcije mokraćnog sustava u ljudi. Nedavno je identificirano nekoliko izolata koji uzrokuju tešku upalu pluća u čovjeka sličnu onoj kod bedrenice. Ti izolati većinom nose virulentni plazmid pXO1 vrste B. anthracis. U ovom radu određena su obilježja triju kliničkih izolata vrste B. cereus izdvojenih iz krvi sadržane u srcu i uzoraka slezene goveda uginulih pod znakovima sličnima bedrenici. Osim određivanja kulturalnih obilježja, pripremljene su i početnice za gen 16S rRNA vrste B. cereus koje su bile rabljene za identifikaciju izolata. Ustanovljeno je da izolati nose plazmid pXO1, a nedostaje im plazmid pXO2. Daljnja karakterizacija plazmida pXO1 pokazala je da izolati sadrže gene pag, lef i cya koji kodiraju za zaštitni antigen, letalni čimbenik i edemski čimbenik, toksine koji su odgovorni za pojavu bolesti u goveda slične bedrenici. Određivanje slijeda i filogenetska analiza dijela sekvencija gena pag izolata B. cereus pokazala je da su oni istovjetni sekvencijama gena pag na pXO1 bakterije B. anthracis. U testu patogenosti na miševima, izolati B. cereus prouzročili su njihovo uginuće šest sati nakon intraperitonejske inokulacije

Sections of brain showing normal architecture of cerebrum and hippocampus in control mice (Hematoxylin and Eosin staining as per the procedure of Luna [17]).

A: Normal appearance of neurons in the cerebrum, B: Normal appearance of neurons in the hippocampus.</p

In vivo titration of KFDV (MLD₅₀) by mice inoculation test.

Each dilution of the KFD seed virus was injected intra-cerebrally to five mice of 3–4 weeks old. Plain medium was injected intra-cerebrally to a group of five mice that served as control group and another five mice were injected with undiluted seed virus that served as positive control. Mice were observed for 14 days at 12-hour intervals for disease symptoms. At the end of 14 days, virus dilutions of 10⁻2, 10⁻3, 10⁻4, 10⁻5 and 10⁻6 caused disease symptoms in 100% of mice, whereas virus dilution of 10⁻7 caused disease in 20% of mice and virus dilution of 10⁻8 did not cause symptoms of the disease in any mice. The control group of mice that received plain medium remained healthy during the study period. All the mice that received undiluted virus showed classical symptoms of KFD.</p

Real-time PCR confirmation of KFD seed virus.

The RNA extracted from the mouse brain suspension containing the KFD seed virus was subjected to Real-time PCR as per the procedure described by Mourya and co-workers [13]. The PCR yielded specific amplifications indicating the presence of KFD virus in the seed stock of mouse brain suspension.</p

The expression levels of Caspase-3 mRNA in KFDV-infected and control mice brain.

The real-time PCR on the RNA extracted from the hippocampus of the KFDV-infected mice brain and control mice brain showed a three-fold increase in Caspase-3 mRNA levels in virus-infected hippocampus compared to the control group (determined by the 2−ΔΔCt method [19, 20]).</p

Determination of MLD₅₀ of the KFD seed virus by i<i>n-vivo</i> titration in mice.

Each dilution of the KFD seed virus was injected intra-cerebrally to five mice of 3–4 weeks old. Plain medium was injected intra-cerebrally to a group of five mice that served as control group and another five mice were injected with undiluted seed virus that served as positive control. Mice were observed for 14 days at every 12 hour intervals for disease symptoms. At the end of 14 days, virus dilutions of 10⁻2, 10⁻3, 10⁻4, 10⁻5, 10⁻6 caused disease symptoms in 100% of mice, whereas virus dilution of 10⁻7 caused disease in 20% of mice and virus dilution of 10⁻8 did not cause symptoms of the disease in any mice. Thus, the one MLD50 of the KFD seed virus (Reed and Muench method) was 10−6.375/0.03 ml.</p

Hind quarter paralysis observed in KFDV-infected mice.

Hind quarter paralysis observed in KFDV-infected mice.</p

Sequential histopathological changes in mice brain following intra-cerebral infection with KFDV (Hematoxylin and Eosin staining as per the procedure of Luna [17]).

A: Day 1- Traumatic hemorrhage in brain, post infection/injection, B: Day 1- Normal appearance of cerebrum, C: Day 1- Normal appearance of hippocampus, D: Day 3- Normal appearance of cerebrum with mild meningeal congestion, E: Day 4- Congestion of meningeal blood vessels, F: Day 5- Meningeal congestion and slow infiltration of inflammatory cells, G: Day 5- Congestion of blood vessels in the cerebrum, H: Day 5- Congestion and perivascular cuffing in the cerebrum, note almost normal appearing neurons in the adjacent areas, I: Day 5- Mild perivascular cuffing and occasional neuronal cell necrosis in the cerebrum, J: Day 6- Meningitis with congestion and infiltration of inflammatory cells, K: Day 6- Vascular congestion and perivascular cuffing in cerebrum, L: Day 6- Microglial aggregation around a vessel. Also note vacuolation of neuropil, degenerated and necrotic neurons in the adjacent area, M: Day 6- Vacuolization of neuropil and presence of necrotic (Red arrow) and apoptotic neurons (Yellow arrow), N: Day 6- A large number of apoptotic neurons (arrow) in the cerebrum, O: Day 6- Microglial aggregation, neuropil vacuolation, necrotic and apoptotic neurons in the cerebrum, P: Day 6- A large number of apoptotic neurons (arrow) in the pyramidal layer of the hippocampus, Q: Day 6- Shrunken neurons with condensed cytoplasm and pyknotic nucleus, in the mid-brain, R: Day 6- Condensation of occasional neuron in the Purkinje cell layer of the cerebellum.</p