Asymptomatic endemic Chlamydia pecorum infections reduce growth rates in calves by up to 48 percent.

Intracellular Chlamydia (C.) bacteria cause in cattle some acute but rare diseases such as abortion, sporadic bovine encephalomyelitis, kerato-conjunctivitis, pneumonia, enteritis and polyarthritis. More frequent, essentially ubiquitous worldwide, are low-level, asymptomatic chlamydial infections in cattle. We investigated the impact of these naturally acquired infections in a cohort of 51 female Holstein and Jersey calves from birth to 15 weeks of age. In biweekly sampling, we measured blood/plasma markers of health and infection and analyzed their association with clinical appearance and growth in dependence of chlamydial infection intensity as determined by mucosal chlamydial burden or contemporaneous anti-chlamydial plasma IgM. Chlamydia 23S rRNA gene PCR and ompA genotyping identified only C. pecorum (strains 1710S, Maeda, and novel strain Smith3v8) in conjunctival and vaginal swabs. All calves acquired the infection but remained clinically asymptomatic. High chlamydial infection associated with reduction of body weight gains by up to 48% and increased conjunctival reddening (P<10(-4)). Simultaneously decreased plasma albumin and increased globulin (P<10(-4)) suggested liver injury by inflammatory mediators as mechanisms for the growth inhibition. This was confirmed by the reduction of plasma insulin like growth factor-1 at high chlamydial infection intensity (P<10(-4)). High anti-C. pecorum IgM associated eight weeks later with 66% increased growth (P = 0.027), indicating a potential for immune protection from C. pecorum-mediated growth depression. The worldwide prevalence of chlamydiae in livestock and their high susceptibility to common feed-additive antibiotics suggests the possibility that suppression of chlamydial infections may be a major contributor to the growth promoting effect of feed-additive antibiotics

Host adaptation of Chlamydia pecorum towards low virulence evident in co-evolution of the ompA, incA, and ORF663 Loci.

Chlamydia (C.) pecorum, an obligate intracellular bacterium, may cause severe diseases in ruminants, swine and koalas, although asymptomatic infections are the norm. Recently, we identified genetic polymorphisms in the ompA, incA and ORF663 genes that potentially differentiate between high-virulence C. pecorum isolates from diseased animals and low-virulence isolates from asymptomatic animals. Here, we expand these findings by including additional ruminant, swine, and koala strains. Coding tandem repeats (CTRs) at the incA locus encoded a variable number of repeats of APA or AGA amino acid motifs. Addition of any non-APA/AGA repeat motif, such as APEVPA, APAVPA, APE, or APAPE, associated with low virulence (P<10-4), as did a high number of amino acids in all incA CTRs (P = 0.0028). In ORF663, high numbers of 15-mer CTRs correlated with low virulence (P = 0.0001). Correction for ompA phylogram position in ORF663 and incA abolished the correlation between genetic changes and virulence, demonstrating co-evolution of ompA, incA, and ORF663 towards low virulence. Pairwise divergence of ompA, incA, and ORF663 among isolates from healthy animals was significantly higher than among strains isolated from diseased animals (P≤10-5), confirming the longer evolutionary path traversed by low-virulence strains. All three markers combined identified 43 unique strains and 4 pairs of identical strains among all 57 isolates tested, demonstrating the suitability of these markers for epidemiological investigations

Multifactorial modeling of <i>C. pecorum</i> infection and host response by principal component and cluster analyses.

<p>Principal component analysis of the 255 observations of 51 calves from week 7 to 15 used the three parameters that best accounted for data variance: plasma levels of anti-<i>C. pecorum</i> IgM antibody, albumin, and globulin. More than 81% of variance observed among animals and between sampling points were explained only by two principal components that were termed to reflect the biological significance of the combination of the original variables as shown with their partial r² in the upper panel. Cluster analysis of PCs for each data point separated all data into two clusters based on Euclidean distances between data points (cluster 1, n = 84; cluster 2, n = 171).</p

Development of calves and chlamydial infection.

<p>The progression over the sampling period is shown (n = 51). (A) Body weight gain over successive 2-week periods and absolute body weight. (B) Conjunctival inflammation as expressed by an arbitrary score from 1–4 for redness, with 2 for normal pink coloration of the conjunctiva. (C) Average <i>C. pecorum</i> genomes per cytobrush swab detected by <i>Chlamydia</i> spp. 23S rRNA gene real-time FRET PCR. Early, midpoint, or late indicates peak <i>C. pecorum</i> infection before week 9, in week 9 or 11, or in week 13 or 15. For clarity, only the mean of all calves is shown with error bars. (D) Anti-<i>C. pecorum</i> IgM antibodies as determined by chemiluminescent ELISA using a lysate antigen of <i>C. pecorum</i> elementary bodies. Data are shown as means ± SEM.</p

Modeling of metabolic health in dependence of earlier anti-<i>C. pecorum</i> immunity.

<p>Predictive modeling by principal component and cluster analysis of plasma albumin and globulin in week 15 of each animal combined with the corresponding anti-<i>C. pecorum</i> IgM 8 weeks earlier in week 7 generated two PCs with a combined r² of 0.82. Two clusters of calves were termed low (n = 34) or high responders (n = 17) based on the values for each PC.</p

Growth, health, and chlamydial infection parameters based on modeling of metabolic health in dependence of earlier anti-<i>C. pecorum</i> immunity.

<p>The progression of low (n = 34) and high responder calves (n = 17) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044961#pone-0044961-g003" target="_blank">Fig. 3</a> over the sampling period from week 7 to week 15 is shown. (A) Body weight gains. (B) Absolute body weight. (C) Plasma insulin-like growth factor-1. (D) Conjunctival inflammation. (E) Anti-<i>C. pecorum</i> IgM. (F) <i>C. pecorum</i> genomes per swab. (G) Plasma albumin. (H) Plasma globulin. For evaluation of statistical significances of differences between responders at sampling time points, data are shown ±95% confidence interval. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Physiological and chlamydial infection parameters categorized by PCA cluster.

a<p>Data are shown ±95% confidence interval. Differences between cluster 1 and 2 are significant at <i>P</i> = 0.0017 for plasma iron, and at <i>P</i><10⁻⁴ for all other parameters.</p

Unrooted neighbor-joining phylogram of <i>ompA</i> of 57 <i>C. pecorum</i> strains based on the nucleotide sequence alignment.

<p>Percentages of branching patterns in bootstrap analyses of the dataset (10,000 replications) are indicated left to the branches. Host animal species, disease association, country of origin, and <i>ompA</i> phylogenetic rank are indicated in the columns to the right of the strain names.</p

Sequence coding tandem repeat characteristics and accession numbers for all <i>C. pecorum</i> strains analyzed in this study.

<p>*Strains sequenced in this study. Strains not marked with an asterisk were sequenced in a preceding study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Salinas1" target="_blank">[23]</a>, or posted as complete genomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Polkinghorne1" target="_blank">[30]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Sait1" target="_blank">[32]</a>.</p>a<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Mojica1" target="_blank">[31]</a>.</p>b<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Kaltenboeck1" target="_blank">[2]</a>.</p>c<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Norton1" target="_blank">[42]</a>.</p>d<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Polkinghorne1" target="_blank">[30]</a>.</p>e<p>Isolated by M. Dawson, Virology Department, Central Veterinary Laboratory, Weybridge UK.</p>f<p>Isolated at INRA, UR1282, Infectiologie Animale et Santé Publique, Centre de Recherche de Tours, France.</p>g<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Rodolakis2" target="_blank">[43]</a>.</p>h<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Sait1" target="_blank">[32]</a>.</p>i<p>Referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103615#pone.0103615-Kaltenboeck2" target="_blank">[3]</a>.</p>j<p>Supplied by Konrad Sachse, Friedrich-Loeffler-Institut Jena, OIE and National Reference Laboratory for Chlamydiosis, 07743 Jena, Germany, 07743 Jena, Germany.</p>k<p>Supplied by Simone Magnino, Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, National Reference Laboratory for Animal Chlamydioses, Sezione Diagnostica di Pavia, 27100 Pavia, Italy.</p>l<p>Isolated by M.S. McNulty, Veterinary Research Laboratory, Stormont, Belfast, Ulster.</p><p>-not amplified by PCR.</p