<i>In Vitro</i> and <i>In Vivo</i> Infectious Potential of <i>Coxiella burnetii</i>: A Study on Belgian Livestock Isolates

<div><p>Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen <i>Coxiella burnetii</i>. Since its discovery, and particularly following the recent outbreaks in the Netherlands, <i>C. burnetii</i> appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of <i>C. burnetii</i> was compared to a reference strain (Nine Mile) using both <i>in vitro</i> (human HeLa and bovine macrophage cells) and <i>in vivo</i> (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to <i>C. burnetii</i>. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. <i>In vitro</i>, the bovine <i>C. burnetii</i> isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.</p></div

Minimum spanning tree analysis of MLVA profiles from Belgian livestock clinical samples positive for <i>C. burnetii</i>.

<p>10-locus MLVA profiles were determined for 11 goat and 20 bovine positive samples collected between 2009 and 2011. Clustering by minimum spanning tree was performed with Bionumerics and included the Nine Mile reference strain and CbNL01 (MLVA profiles derived from publicly available data). Circles outline the genetic profiles of strains found in positive clinical samples. Numbers on the connecting lines refer to the number of markers differing between samples. The size of the circles is proportional to the number of strains bearing the same genetic profile. Most bovine strains (19/20) are closely related. One goat sample (CAP3) was infected with a strain displaying a genetic profile identical to that of CbNL01.</p

Growth curves of NMI, CbBEB1 and CbBEC1 in HeLa and SV40 cells.

<p>Cells were infected at 100 M.O.I. for 24 hours. Infected cells were then washed and grown in gentamicin-containing medium for 96 additional hours. At fixed time-points, deionized water was added and cells lysed by a 30-min incubation at 82°C. <i>C. burnetii</i> loads were quantified by qPCR on 1∶15 dilutions. Each point indicates the average of four independent cultures and its corresponding standard deviation.</p

<i>C.burnetti</i> colonization of spleen and lungs as evaluated by qPCR quantification and organ weight.

<p>Graphs show <i>C. burnetii</i> loads in spleen and lungs (left panels) or variations in spleen and lungs weight (right panels). Results are given for uninfected mice and mice infected with NMI, CbBEB1 and CbBEC1 at 1, 2, 4 and 8 weeks post infection. Mean values +/− Standard Error (SE) for 5 mice are shown. * indicates p-values smaller than 0.05 ** indicates p-values smaller than 0.01.</p

Mice anti-<i>C. burnetii</i> IgM and IgG assayed by ELISA at various time-points.

<p>IgM (left panel) and IgG (right panel) average levels of anti-<i>C. burnetii</i> antibodies as measured by ELISA in NMI, CbBEB1 and CbBEC1 infected mice at 1, 2, 4 and 8 weeks post-infection. Optical density values (O.D.) were normalized with values from uninfected control mice. Each value shown is the mean value determined on 5 mice +/− SE.</p

List of <i>C. burnetii</i> isolates used in this study.

<p>country of origin, source of isolation, SNP and MLVA genotypes. The goat-derived strain (CbBEC1) has been isolated from an animal imported from the Netherlands. This strain has the same genetic profile as the CbNL01, the <i>C. burnetii</i> founder strain of the 2007–2010 Dutch outbreak episodes. SNP and MLVA profile of CbNL01 was derived from publicly available data.</p>*<p>approximated value.</p

Sequence analysis of the <i>RpoB</i>-gene.

<p>Sequence analysis of the <i>RpoB</i>-gene.</p

Detection and characterization of <i>Brucella</i> spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan

<div><p>Brucellosis is one of the most common zoonoses globally, and Central Asia remains a <i>Brucella</i> hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as <i>Brucella</i>. This study investigates the presence of <i>Brucella</i> DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. <i>Brucella</i> DNA was detected in 10.3% of 564 cow milk samples by IS<i>711</i>-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, <i>Brucella</i> DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of <i>Brucella</i> DNA. In addition, sequence analysis of the <i>rpoB</i> gene suggests that one cow was infected with <i>B</i>. <i>abortus</i> and another cow was most likely infected with <i>B</i>. <i>melitensis</i>. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in <i>Brucella</i> surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control <i>Brucella</i> infections in the livestock population but further research focusing on the isolation of <i>Brucella</i> is required to obtain a comprehensive understanding of the <i>Brucella</i> spp. circulating among the livestock in this region.</p></div

Ct-values from the IS<i>711</i>-based qPCR of the seronegative individuals (n = 46).

<p>The blue dots represent Ct-values from the first run and the red dots represent Ct-values from the second run.</p

Total number of herds surveyed and animals tested in the urban (Ur), peri-urban (Pu) and rural areas (Ru) of Niger.

<p>Total number of herds surveyed and animals tested in the urban (Ur), peri-urban (Pu) and rural areas (Ru) of Niger.</p