Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known

Experimental setup of the animal study.

<p>Experimental setup of the animal study.</p

Inhibition of bacterial adhesion to intestinal epithelial cells.

<p>Bacteria (<i>S</i>. <i>diarizonae</i>, <i>E</i>. <i>coli</i> O138, F4+ or F18+) fluorescently stained as explained in Materials and Methods, and treated with ppIgG in different concentrations (from 100 mg/ml to 0.2 mg/ml) were incubated with IPEC-J2 cells. Binding of bacteria to IPEC-J2 is depicted in relation to non-inhibited bacteria (no ppIgG, set to 100%). One typical experiment is shown.</p

The impact of natural immunoglobulins on enteral <i>Enterobacteriaceae</i>.

<p><b>(A)</b> Shedding of <i>E</i>. <i>coli</i> F4+ in an <i>in vivo</i> infection model. Animals were infected orally at the day of weaning as well as the day after weaning with <i>E</i>. <i>coli</i> F4+ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147373#sec002" target="_blank">Materials and Methods</a>), after which faecal shedding was followed with a F4 specific qPCR. Data were log-transformed before analysis by the non-linear equation, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147373#sec002" target="_blank">Materials and Methods</a> for further details. <b>(B)</b> Ileal content of family <i>Enterobacteriaceae</i>. Bacteria were enumerated by NGS sequencing of the 16S rDNA gene in ileal samples obtained at necropsy by the end of the experiment. Values are normalized (per 100,000 reads) and log-transformed read counts. Overall ANOVA p-value < 0.001, different letters denote significantly different values.</p

Characterisation of ppIgG.

<p><b>(A)</b> Pig plasma IgG product (ppIgG, 1 mg/ml) was run under reducing conditions on 12% SDS PAGE as described in Materials and Methods. In parallel, Western blotting (WB) was performed on the same sample. The blot was developed with biotinylated rabbit anti-pig IgG F(ab)₂ antibody, followed by alkaline phosphatase-coupled streptavidin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147373#sec002" target="_blank">Materials and Methods</a>). <b>(B)</b> Wells were coated with formalin-fixed bacteria (Y. ruckeri, S. diarizonae, <i>E. coli</i> O138, O149:F4 and F18+) in separate ELISA plates, and incubated with ppIgG added in serial 2-fold dilutions, and the bound IgG was estimated by using a HRP-conjugated rabbit anti-pig IgG antibody. Data from one typical experiment are presented. <b>(C)</b> ppIgG was tested for reactivity against <i>Escherichia coli</i> and <i>Salmonella diarizonae</i> by competitive ELISA as described (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147373#sec002" target="_blank">Materials and Methods</a>). ppIgG heated at 60°C or 70°C for 1 hour and PBS were used as control samples. The results are presented as ‘% inhibition’ of the signal obtained in the absence of ppIgG (median values (± median ranges)). Two-way ANOVA (subjected to Bonferonni post-test) was used to observe statistical significance between the treated and untreated ppIgG (***: p<0.01; **: p<0.01).</p

The relative composition of the ileal microbiota on a family level.

<p>Bacteria were enumerated by NGS sequencing of the 16S rDNA gene in ileal samples obtained at necropsy by the end of the experiment.</p