Assessment of Histopathological Changes in the Liver and Spleen of Mice Infected with Salmonella typhi Following Treatment with Ciprofloxacin

This study evaluated the toxicity of ciprofloxacin to spleen and liver when used for the treatment of mice infected with S. typhi for seven days. The dose concentration used in these experiments was 100mg/kg. Mice were divided into two groups . The first group (negative control) was not given ciprofloxacin, but rather a sterile phosphate buffer solution (PBS) as an alternative. Ciprofloxacin was administered to the second group. After seven days , the animals were sacrificed and organs (liver and spleen) were collected . The histopathological examination showed normal hepatocytes in the liver and normal structure of spleen cells in animals of control group . However, the treated group showed dilated and congested blood vessels with perivascular inflammatory cell cuffing and acute cell swelling in the liver, as well as white pulp activation with an increased number of megakaryocyte cells in the spleen. Therefore, the current study suggests that the concentration of 100 mg/kg of ciprofloxacin is considered to be toxic to hepatocytes and splenocytes of mice during the treatment period.

Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Comparison of three nasopharyngeal swab types and the impact of physiochemical properties for optimal SARS-CoV-2 detection

Adequate swab specimen collection, release and detection of nucleic acids by molecular diagnostic assays is largely attributed to the physical and chemical characteristics of different swab types. We investigated properties of three types of commercial nasopharyngeal swabs (nylon flocked: Type 1-Media Merge; Type 2-Kang Jian Medical Apparatus, China and Type 3-Wuxi NEST Biotechnology Co. Ltd, China) used in clinical diagnostics with the aim to establish if different swab designs and configurations had any effect on swab performance. Properties investigated included viral absorption, release, capture, extraction and recovery efficiency from each swab for the detection of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). All swab types (n=18) were inoculated with different amounts of SARS-CoV-2 live viral cultures (1:10, 1:100 and 1:1000 copies/ml) and eluted in sterile phosphate buffer saline. RNA was extracted from all swab eluates using a fully automated system (BD MAX™ System) and cycle threshold (Ct) values were compared. RNA stability was also investigated after dry storage of swabs at room temperature for 72 hours. Statistically significant differences (p<0.05) were observed in the absorption and release capabilities between Type 1 and 3 as well as between Type 2 and 3 swabs, however, no significant difference was observed between Type 1 and 2. Ct values and extraction efficiency amounts of SARS-CoV-2 varied amongst the swab types. We conclude that in order to facilitate accurate SARS-CoV-2 diagnosis, assessment of NP swab characteristics is of importance before implementation for specimen collection in the clinical setting.

COMPARISON OF THE EFFECT OF ANTIBIOTICS AND BACTERIOPHAGE PHAGE SAVB14 ON BIOFILMS FORMED BY STAPHYLOCOCCUS AUREUS VARIANT BOVIS

During the development of mastitis in cows, the formation of a biofilm pathogen is an effective way to preserve it in the microenvironment of mammary gland. Biofilm infections are difficult to treat with antimicrobials, and bacterial resistance to antibiotics increases to 1000-fold level, compared with what is observed when grown in planktonic conditions. The aim of study – to determine and compare the effect of antimicrobial drugs and bacteriophage Phage SAvB14 in the destruction of biofilms formed by S. aureus var. bovis. Isolation and species identification of staphylococci were performed according to conventional methods using BD Baird-Parker Agar medium (HiMedia, India). Determination of ability of staphylococci to form biofilms and the number of viable bacteria was determined by the Stepanovic method. The study of sensitivity of microorganisms in biofilm form was performed on daily microbial biofilms grown in plastic Petri dishes. After 24 hours of incubation of cultures, the dishes were washed three times from planktonic (unattached) microorganisms with sterile phosphate buffer and introduced the studied antibacterial agents. After exposure, the dishes were washed three times with sterile phosphate buffer, introduced 5 cm3 of sterile 0.9% sodium chloride solution and washed the biofilm, took 1.0 cm3 of suspension, prepared a series of ten-fold dilutions, inoculated 1.0 cm3 of each dilution in Petri dishes, poured MPA and incubated at temperature of 370C for 24–48 hours to determine the number of bacteria. In determining the effect of antibiotics on bacterial biofilms, it was found that of the studied antibiotics, enrofloxacin worked best probably due to its low molecular weight and ability to penetrate the pores and channels of the biofilm to microbial cells. After the action of enrofloxacin on biofilms, staphylococcal bacteria were completely inactivated. Also, the antibiotics ceftriaxone and doxycycline were effective against bacteria in biofilms. After the action of ceftriaxone, the number of surviving bacteria was lg 1.9 ± 1.1 CFU/cm2 of biofilm area, and doxycycline lg 2.5 ± 1.2 CFU/cm2. At the same time, under the action of antibiotics penicillins, aminoglycosides and macrolides, the number of surviving microbial cells was about lg 5.3 CFU/cm2 of biofilm area. In studies on the effect of bacteriophage Phage SAvB14 on biofilms formed by S. aureus var. bovis, there was their degradation. At this, viable microbial cells from the biofilm were not isolated. In this case, we can say that the phages penetrated and reached the staphylococcal cells throughout the thickness of biofilm and bacteria were susceptible to this phage. That is, there was a passive treatment of biofilm with phages, in which lysis depended on the rate of virus uptake. Therefore, the obtained results of laboratory studies indicate the prospects of effective use of our selected specific staphylococcal bacteriophage Phage SAvB14 for the destruction of biofilm formed by S. aureus var. bovis – in mastitis of cows.

Pathological changes of immunized mice with Trichophyton mentagrophyte lyophilized antigen

The study was carried to investigate the pathological effect of lyophilized antigen of Trichophyton mentagrophytes in mice. Fifty mice were divided into three groups. The first group 20 mice were immunized subcutaneous (s/c) with 0.5 ml of T.mentagrophyte antigen 20 µgm/ml, by two doses, 14 day intervals, between them. The second group 20 mice and third group 10 mice considered as positive and negative control groups respectively. After 30 days post immunization first and second groups were challenged intradermal I/d. with 0.1 ml of fungal suspension contain (1×107 ml) of viable virulence T.mentagrophyte while the third group injected intraperitoneally I/P. with 0.5 ml of sterile phosphate buffer saline. All mice of the first and second groups were sacrificed at (5, 14, 30 and 60) days post challenge for gross and histopathological examination. Histopathologically the second group showed epidermal hyperkeratosis with appearance of crust lesions seen with abscess formation especially in early stage of lesion, while the main feature of advance cases were characterized by folliculitis with fungal hyphae invasion in all epidermal and dermal layers together with eosinophilic infiltration. Mild pathological changes were seen in the 1st immunized group characterized by infiltration of mononuclear cells mainly macrophages in dermal connective tissue with dense proliferation of collagenous fibers , appearance of young fibroblasts together with cellular hypodermal infiltration of eosinophil no evidence of clear follicular lesions were seen. Lyophilized antigen of T.mentagrophyte can be considered as an effective immunogen for protecting mice against T.mentagrophyte infection and it is synchronized with its dose.

Sealing Properties of Three Luting Agents Used for Complete Cast Crowns: A Bacterial Leakage Study

SUMMARY Objective To assess the sealing properties of three different luting materials used for cementation of full cast crowns on extracted human premolars. Methods Thirty noncarious human premolars were prepared in a standardized fashion for full cast crown restorations. All margins were placed in dentin. After impressions of the preparations, stone dies were fabricated on which copings were waxed, which were cast in type III alloy using standardized laboratory methods. Teeth were randomly assigned to three groups of 10 samples each (n=10), for which the following cements were used: 1) a resin-modified glass ionomer cement, Rely X Luting Plus (3M ESPE, St Paul, MN, USA); 2) a self-adhesive resin cement, Maxcem Elite (Kerr Corporation, Orange, CA, USA); and 3) a glass ionomer cement, Ketac Cem (3M ESPE), the latter used as control. After cementation the samples were allowed to bench-set for 10 minutes, stored in water at 37°C, subjected to thermal cycling (2000×, between 5°C and 55°C, dwell time 35 seconds), and then stored in sterile phosphate buffer for seven days at 37°C. Subsequently, the occlusal surface was carefully reduced until the dentin was exposed. Finishing on wet sand paper removed the gold flash caused by grinding. After sterilization, the specimens were subjected to bacterial microleakage in a dual chamber apparatus for 60 days. Bacterial leakage was checked daily. Data were analyzed using the Kaplan-Meier survival test. Significant pairwise differences were analyzed using the log-rank test followed by Fisher exact test at a p&lt;0.05 level of significance. Results Rely X Luting Plus showed the lowest microleakage scores, which statistically differed significantly from Maxcem Elite and Ketac Cem (p&lt;0.05). Conclusions Rely X Luting Plus cement displayed significantly lower microleakage scores than a self-adhesive resin-based and conventional glass ionomer cement.

Experimental pathogenesis of pullorum disease in chicks by local isolate of Salmonella Pullorum in Bangladesh

This study was undertaken to observe the experimental pathogenesis of locally isolated Salmonella enterica subspecies enterica serovar Pullorum in chicks. Fifty chicks were experimentally infected by the oral route with 2 x 107 (CFU) units of Salmonella Pullorum organisms reconstituted in 0.5 ml of sterile phosphate buffer saline (PBS), PH 7.2 and 50 chicks were given only 0.5 ml of sterile PBS as control. Observations were made on clinical signs, gross pathology, and reisolation of S. Pullorum from different organs and blood, histopathological lesions, detection of antibody levels and detection of S. Pullorum by PCR at different time intervals of experimental period. Five birds were randomly selected and sacrificed on 6 hrs before inoculation and 6 hrs, 12 hrs, 24 hrs, 2 days, 3 days, 1st week, 2nd, 3rd and 4th weeks of post infection (PI). The clinical signs of infected chicks were depression, loss of appetite, huddled together, loss of feed and water intake, reduced mean body weights, ruffled feathers, diarrhoea, laboured breathing and pasty vent. The highest gross lesion was (84%) unabsorbed and coagulated yolk and the lowest lesion was (32%) pericarditis and necrotic foci/ nodules in heart. Microscopically, the liver showed congestion, focal necrosis with multifocal infiltration of histiocytes in liver parenchyma. Salmonella organisms were reisolated from different organs and blood at 12 hrs PI. The antibody titre increased gradually and the highest titer was 7275.717 ± 5087.24 at 4 wks PI. In rapid plate agglutination test, the positive result was found from one wk of PI with the sera of infected birds. At 12hrs PI Salmonella was detected by PCR from 20% liver and 20% lung samples of infected birds and no Salmonella was isolated from control group. The orally inoculated Salmonella Pullorum organisms produced lesions in digestive tract, invaded digestive tracts and entered to blood and seeded to different organs in different time intervals and ultimately produced clinical signs, gross and microscopic tissue lesions with immunological response. DOI: http://dx.doi.org/10.3329/jbau.v10i1.12098 J. Bangladesh Agril. Univ. 10(1): 87–94, 2012

Immune relationahip between Pseudomonas aeruginosa and Listeria monocytogenes

In order to known the effect of whole sonicated L. monocytogenes antigen on as mice experimentallyinfected with P.aeruginosa.,30 white mice,8-12 weeks age, were divided randomly into three equalgroups..The 1st group was immunized subcutaneously twice with (0.5)ml of whole sonicated P.aeruginosaantigen(7.6mg/ml concentration protein) ,with two weeks intervals.The cellular immune responses waschecked at (27) days post-immunization The animals of the 1st and 2nd groups were challengedsubcutaneously with (0.5) ml of bacterial suspension contain1X10 9cfu/ml of virulent P.aeruginosa whilethe 3rd group was inoculated S/C with (0.5)ml of sterile phosphate buffer saline and served as controlnegative group.. The resuls showed that the prepared antigen antigen induced a goodprotection in the immunized animals which was,characterized by survival all immunized animals and nobacterial isolates and pathological changes in the internal organs after infection with P.aeruginosa ascompared with nonimmunized infected animals,which died after infection, with severe bacterial isolatesand sever acute suppurative reaction in their internal organs

Phytic Acid Exposure Alters AflatoxinB1-Induced Reproductive and Oxidative Toxicity in Albino Rats (Rattus norvegicus)

The increased use of feed in Egypt's aquaculture and animal industries raises concerns about the possible presence of mycotoxins in feedstuffs. The use of alternative medicine, such as botanicals and nutritional supplements, has become popular with inflammatory cases. The present study aimed to testify the role played by phytic acid (IP6) in enhancing the reproductive and oxidative toxicity induced in aflatoxinB1 (AFB1) treated white male albino rats (Rattus norvegicus) throughout treatment and withdrawal periods. One hundred and twenty white male albino rats were grouped into four groups. Group 1, was injected with 300 μg kg−1body wt of AFB1 once every 3 days for 15 days and left uninjected for another 15 days to study the withdrawal effect. Group 2, was injected with 300 μg kg−1body wt of AFB1 once every 3 days for 15 days and treated simultaneously with IP6 daily for another 15 days. Group 3, was treated daily with IP6 (40 mg kg−1body wt) for 15 days and with no treatment for other 15 days. Group 4, injected with equivalent volume of sterile phosphate buffer saline solution as a control group. Sera were taken at the experimental intervals and assayed for testosterone hormone, follicular-stimulating hormone (FSH) and luteinizing hormone (LH) to determine the toxicological impact of AFB1 and the possibility of amelioration by phytic acid on the reproductive performance of the studied animal. The effects of AFB1 treatment on the absolute and relative weight of testis as well as its histopathologic effect on the testis and the possibility of amelioration by IP6 treatment were evaluated. The activities of enzymatic and non-enzymatic anti-oxidants, in addition to lipid peroxidation were measured in the testis’ homogenate of AFB1-treated rats. A decrease in sex hormone levels, an increase in testicular lipid peroxidation product levels and a significant decrease in testicular glutathione content, catalase and total peroxidase and superoxide dismutase activities were recorded. The histopathologic alterations revealed a degeneration and highly mitotic division within the spermatogenic nuclei, in addition to some karyomegaly and nuclear pyknosis. It is concluded that the reduction in the toxicity of free radicals by phytic acid might be responsible for the protective influence observed.

First Report of Bacterial Blight on Conventionally and Organically Grown Arugula in Nevada Caused by Pseudomonas syringae pv. alisalensis

In 2007, leaf spots were observed on arugula (Eruca vesicaria subsp. sativa cv. My Way) grown under sprinkler irrigation for fresh market in conventional and organic production fields located above 1,200 m (4,000 feet) in Nevada (NV). Approximately 30% of each planting was affected. Initially, symptoms consisted of small (<2 mm in diameter), angular, water-soaked spots visible from both sides of the leaf, some of which developed a shot-hole appearance. The spots enlarged and coalesced, remaining angular. Lesions ranged from black to tan, occasionally developing a chlorotic or purple margin. Some lesions resembled symptoms of downy mildew on arugula, but microscopic examination revealed no sporangiophores associated with the lesions. Bacterial ooze was observed when sections of symptomatic leaves were examined microscopically. Blue-green fluorescent pseudomonads were isolated from lesions on King's medium B agar from three arugula plantings. Twelve strains (at least three from each planting) were evaluated along with known strains of Pseudomonas syringae pv. alisalensis and P. syringae pv. maculicola in all assays. Bacterial strains were levan positive, oxidase negative, and arginine dihydrolase negative. Strains did not rot potato slices but induced a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv. Sansun) indicating that the bacteria belonged to Lelliot's LOPAT group 1 (2). This was confirmed by analysis of fatty acid methyl esters (MIS-TSBA version 4.10; MIDI, Inc., Newark, DE), which indicated that the strains were highly similar (similarity >0.80) to P. syringae. Amplification of DNA between repetitive bacterial sequences (rep-PCR) using the BOXA1R primer resulted in identical banding patterns for the NV arugula strains and P. syringae pv. alisalensis from arugula in California (1). Koch's postulates were completed by confirming pathogenicity of the isolated strains on the arugula cvs. Italian and Astro. Strains were grown on nutrient agar for 48 h at 27°C, adjusted to 108 CFU/ml in sterile 0.01 M phosphate buffer (pH 7.0), and spray inoculated until runoff onto 2- to 3-week-old plants. Control plants were similarly sprayed with sterile phosphate buffer. Plants were held for 2 days in a mist chamber and 7 days on a greenhouse bench (24 to 26°C). Angular lesions similar to symptoms observed on the original plants developed on leaves of all inoculated arugula plants. In addition, some plants developed blackening of the smaller veins accompanied by chlorosis of the surrounding interveinal tissue in 10- to 20-mm diameter areas of the leaves. Small black lesions (as much as 10 mm long) were also observed on the petioles. Bacterial strains reisolated from the symptomatic tissue were identical to P. syringae pv. alisalensis by rep-PCR. Control plants remained symptomless. Similar inoculation and incubation methods confirmed that the host range of the NV arugula isolates was identical to that of known strains of P. syringae pv. alisalensis. The arugula and P. syringae pv. alisalensis isolates caused leaf spots on broccoli raab (Brassica rapa subsp. rapa cv. Sorento) and oats (Avena sativa cv. Montezuma). Pathogenicity tests were repeated. This confirms that the leaf spot observed on conventionally and organically grown arugula in NV was caused by P. syringae pv. alisalensis. To our knowledge, this is the first report of this disease on arugula in NV. References: (1) C. T. Bull et al. Plant Dis. 88:1384, 2004. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.