Polymerase chain reaction for the identification of bacteria.

<p>Genomic DNA was isolated from the obtained isolates as well as reference strains, and subjected to mono- or multi-plex PCR as described in the Materials and Methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142717#pone.0142717.t001" target="_blank">Table 1</a>. The experiments were repeated at least three times and representative gel pictures are shown. Note that each panel is composed from two separate gels since all the samples could not be accommodated in a single gel. <b>(A) PCR for genus-specific <i>tuf</i> genes of streptococci and staphylococci.</b> Lane designation: M, 100 bp ladder; 1–5, <i>Streptococcus</i> spp. isolates; 6, Reference strain Streptococcus AD1; 7, No template control for streptococcus; 8, Negative control (<i>S</i>. <i>aureus</i>, <i>E</i>. <i>coli</i>); 9, Reagent control; 10, Reference strain <i>S</i>. <i>aureus</i> 96; 11, No template control for staphylococcus; 12–18: <i>Staphylococcus</i> spp. isolates. <b>PCR for <i>S</i>. <i>aureus nuc</i> (lanes 1–11) and <i>E</i>. <i>coli alr</i> (lanes 12–21) genes.</b> Lane designation: M, 100 bp ladder; 1–8, <i>S</i>. <i>aureus</i> test isolates; 9, Reference strain SAU-3; 10, Negative control (<i>E</i>. <i>coli</i>); 11, No template control; 12, Negative control (<i>S</i>. <i>aureus</i>); 13, Reference strain EC11 (<i>E</i>. <i>coli</i>); 14–16, Test isolates of <i>E</i>. <i>coli</i>; 17, No template control; 18–20, Test isolates; 21, Negative control (streptococcus). <b>(B) PCR for the identification of CoNS species.</b> Lane designation: M, 100 bp ladder; 1, <i>S</i>. <i>haemolyticus</i> (MTCC 3383) control; 2, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 3, <i>S</i>. <i>saprophyticus</i> (MTCC 6155) control; 4, <i>S</i>. <i>arlettae</i> (JQ764624) control; 5, <i>S</i>. <i>chromogenes</i> (MTCC 3545) control; 6, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 7, <i>S</i>. <i>xylosus</i> (FJ90627.1) control; 8, <i>S</i>. <i>simulans</i> (AF495498.1) control; 9, <i>S</i>. <i>epidermidis</i> (MTCC 3615) control; 10, <i>S</i>. <i>haemolyticus</i> (MTCC 3383) control; 11, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 12, <i>S</i>. <i>saprophyticus</i> (MTCC 6155) control; 13, <i>S</i>. <i>arlettae</i> (JQ764624) control; 14, <i>S</i>. <i>chromogenes</i> (MTCC 3545) control; 15, <i>S</i>. <i>sciuri</i> (MTCC 6154) control; 16, <i>S</i>. <i>simulans</i> (AF495498.1) control; 17, <i>S</i>. <i>xylosus</i> (FJ90627.1) control; 18, <i>S</i>. <i>epidermidis</i> (MTCC 3615) control. This Panel represents two mutually exclusive pictures depicting the results of the standardization of one tube each of the two-tube multiplex PCR. In the left panel, primers for <i>S</i>. <i>arlettae</i>, <i>S</i>. <i>chromogenes</i>, <i>S</i>. <i>sciuri</i>, <i>S</i>. <i>epidermidis</i> and <i>S</i>. <i>saprophyticus</i> were used, and <i>S</i>. <i>haemolyticus</i>, <i>S</i>. <i>xylosus</i> and <i>S</i>. <i>simulans</i> DNA served as negative controls. In the right panel, primers for <i>S</i>. <i>equorum</i>, <i>S</i>. <i>haemolyticus</i>, <i>S</i>. <i>xylosus</i>, <i>S</i>. <i>simulans</i> and <i>S</i>. <i>fluerettii</i> were used, and <i>S</i>. <i>sciuri</i>, <i>S</i>. <i>sapryphyticus</i>, <i>S</i>. <i>arlettae</i>, <i>S</i>. <i>chromogenes</i> and <i>S</i>. <i>epidermidis</i> DNA served as negative controls. Numbers in parentheses indicate the GenBank Accession numbers or the MTCC culture designations. <b>(C) PCR for the identification of <i>Streptococcus</i> species.</b> Lane designation: M, 100 bp ladder; 1–20, Test streptococcal isolates streptococci (no amplification); 21, Negative control (<i>S</i>. <i>aureus</i>); 22, Negative control (<i>E</i>. <i>coli</i>); 23 & 24, No template control; 25, Tube 2 positive control (<i>Streptococcus</i> reference strain AD3); 26, Tube 1 positive controls (<i>Streptococcus</i> reference strains AD1 and AD6).</p

Detection of IL-1β and TNF-α levels in serum samples of mice 48 h post-infection.

<p>Detection of IL-1β and TNF-α levels in serum samples of mice 48 h post-infection.</p

Histopathological changes observed in mouse mammary tissue.

<p>Control mouse showing absence of inflammatory response (Level 0) in mammary tissue (A) to inoculation of sterile normal saline, H&E x100, bar, 200 μm (Fig 1a); Level 1 inflammatory response induced in the mouse teat by inoculation of <i>S</i>. <i>aureus</i> via the mammary duct (A), characterized by acute neutrophil rich response in the intraductal exudate, H&E x400, bar, 50 μm (Fig 1b); Level 2 inflammation of the mammary glands (A) Infiltrate of acute inflammatory cells, predominantly neutrophils, in supporting connective tissue, with intraluminal organisms, H&E x200, bar, 100 μm (Fig 1b); Level 2 inflammation of the mammary tissue post-inoculation of mammary gland with <i>S aureus</i>, characterized by infiltration of acute inflammatory cells, predominantly neutrophils (A), in supporting connective tissue and intraluminal space, H&E x200, bar, 100 μm (Fig 1c); Level 3 inflammation of mammary tissue post-inoculation of mammary gland with <i>S</i>. <i>aureus</i>, characterized by marked acute neutrophil rich infiltrate (A) with tissue necrosis (B), H&E x200, bar, 100 μm (Fig 1c); Level 3 inflammation of the mammary tissue post-inoculation of mammary gland with <i>S</i>. <i>aureus</i>, characterized by marked acute neutrophil-rich infiltrate (A) with tissue necrosis (B), H&E x200, bar, 100 μm (Fig 1d); Level 3 inflammation of the mammary tissue post-inoculation of mammary gland with <i>S</i>. <i>aureus</i>, characterized by neutrophil-rich inflammatory exudate, H&E x1000, bar, 20 μm (Fig 1e); Level 3 inflammation of the mammary tissue post-inoculation of mammary gland with <i>S</i>. <i>aureus</i>, characterized by presence of Gram-positive bacteria and neutrophil-rich inflammatory exudate, Gram Twort x400, bar, 50 μm (Fig 1f).</p

Phenotypic and genotypic characteristics of <i>S</i>. <i>aureus</i> strains used in this study.

<p>Phenotypic and genotypic characteristics of <i>S</i>. <i>aureus</i> strains used in this study.</p

Clinical signs observed in mice infected with strong versus weak-biofilm forming <i>S</i>. <i>aureus</i> strains by intra-mammary route (observations up to 48 hours post inoculum).

<p>Clinical signs observed in mice infected with strong versus weak-biofilm forming <i>S</i>. <i>aureus</i> strains by intra-mammary route (observations up to 48 hours post inoculum).</p

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

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

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

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

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