101 RISK OF COXIELLA BURNETII TRANSMISSION BY EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 165
Author(s):  
A. Alsaleh ◽  
J. L. Pellerin ◽  
D. M. Garcia ◽  
D. Tainturier ◽  
F. Fieni
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Economic Losses ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Worldwide Distribution ◽  
Embryo Cells ◽  
Means Of Transmission

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Domestic ruminants are the main sources of infection for humans. In cattle, infection is frequently asymptomatic, but it may cause abortion, reproductive failure (metritis, placentitis, and infertility), and economic losses. A previous study in goats showed that Coxiella burnetii had a strong tendency to cling to the zona pellucida (ZP) after in vitro infection and the washing procedure recommended by IETS for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology). The aims of this study were to determine (1) whether Coxiella burnetii would adhere to the intact ZP (ZP-intact) of early in vitro-produced bovine embryos, (2) whether the bacteria would adhere to or infect the embryo cells (ZP-free) after in vitro infection, and (3) the efficiency of the washing protocol recommended by the IETS. One hundred and sixty 8- to 16-cell bovine embryos produced in vitro were randomly divided into 16 batches of 10 embryos each. Twelve batches (8 ZP-intact and 4 ZP-free) were incubated in medium containing C. burnetii CbB1 (IASP, INRA Tours, France). After 18 h of incubation at 37°C and 5% CO2 in air, the embryos were washed in 10 successive baths of a phosphate buffer saline (PBS) and 5% FCS solution in accordance with the IETS guidelines. In parallel, 4 batches (2 ZP-intact and 2 ZP-free) were subjected to similar procedures but without exposure to C. burnetii to act as controls. The 10 washing fluids for all batches were collected and centrifuged for 1 h at 13 000 × g. Embryo and pellet washing were tested by C-PCR. Coxiella burnetii DNA was found in all ZP-intact and ZP-free embryo batches after 10 successive washes. It was also detected in the first 4 washing fluids for ZP-intact embryos and in the 10th washing fluid for 2 of the 4 batches of ZP-free embryos. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. burnetii adhere and (or) penetrate the early embryonic cells as well as the ZP of in vitro bovine embryos after in vitro infection and the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring, or both. Further studies are needed to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?

2013 ◽  
Vol 25 (1) ◽  
pp. 231
Author(s):  
A. Alsaleh ◽  
J. L. Pellerin ◽  
C. Roux ◽  
M. Larrat ◽  
G. Chatagnon ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Early Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Tissue Samples ◽  
Worldwide Distribution

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.

162 CAN CHLAMYDIA ABORTUS BE TRANSMITTED BY EMBRYO TRANSFER IN GOATS?

2015 ◽  
Vol 27 (1) ◽  
pp. 172
Author(s):  
J. L. Pellerin ◽  
A. Ashraf ◽  
M. Oseikria ◽  
K. Laroucau ◽  
F. Vorimore ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Chlamydia Abortus ◽  
Zoonotic Risk ◽  
Embryo Cells ◽  
Means Of Transmission ◽  
Infectious Form

Chlamydia abortus is a gram-negative obligate intracellular bacterium. Its lifecycle includes a resistant infectious form and a metabolically active non-infectious form. Chlamydia abortus infection results in abortion in goats; in nonpregnant animals the infection is usually subclinical. Chlamydia abortus presents a major zoonotic risk for pregnant women. The aim of this study was to investigate whether the embryonic zona pellucida (ZP) protects early embryo cells from infection and to test the efficacy of the washing protocol recommended by the IETS for bovine embryos. The study was performed in triple replicate: 14 donor goats, certified negative by ELISA and PCR to C. abortus, were synchronized, superovulated, and subsequently inseminated by males controlled negative for C. abortus. Fifty-two ZP-intact embryos (8–16 cells) were collected 4 days later, by laparotomy. The embryos were randomly divided into 12 batches. Nine batches of 5 embryos were incubated in a medium containing 4 × 107 Chlamydia mL–1, AB7 strain. After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of PBS and 5% FCS solution in accordance with IETS guidelines for bovine embryos. In parallel, 3 batches of ZP-intact embryos (2, 2, and 3 embryos in the first, second, and third batches, respectively) were used as controls by being subjected to similar procedures, but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 h at 13 000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at –20°C before examination for evidence of C. abortus using RT-PCR. Chlamydia abortus DNA was found in all batches of infected ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the tenth wash fluid for 4 batches (4/9) of infected embryos. As expected, none of the embryos or their washing fluids in the control batches were DNA positive. These results demonstrate that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goat to healthy recipients and/or their offspring. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of infected caprine embryos can eliminate C. abortus from the ZP.

Risk of Coxiella burnetii transmission via embryo transfer using in vitro early bovine embryos

2014 ◽  
Vol 81 (6) ◽  
pp. 849-853 ◽  
Author(s):  
A. Alsaleh ◽  
F. Fieni ◽  
D. Moreno ◽  
E. Rousset ◽  
D. Tainturier ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Coxiella Burnetii ◽  
Bovine Embryos

111 RISK OF CHLAMYDIA ABORTUS TRANSMISSION VIA EMBRYO TRANSFER USING IN VITRO EARLY BOVINE EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 186
Author(s):  
F. Fieni ◽  
M. Oseikria ◽  
K. Laroucau ◽  
F. Vorimore ◽  
D. Tainturier ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Early Embryo ◽  
Control Group ◽  
Bovine Embryo ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Chlamydia Abortus ◽  
Zoonotic Risk

Chlamydia abortus (C. abortus) in cattle has been reported sporadically throughout the world and is implicated in respiratory, ocular, and reproductive disease as abortion, infertility, chronic mastitis, vaginal discharge, and endometritis. In addition, C. abortus presents a zoonotic risk exposure of pregnant women to infected animal and can lead to severe septicaemia in the mother, resulting in spontaneous abortion or stillbirth of the fetus. To investigate the risk of C. abortus transmission via bovine embryo transfer, our study aims to determine whether the embryonic ZP of in vitro-produced embryos protects early embryo cells against C. abortus infection and whether the bacteria adhere to or infect the cells of early bovine embryos (ZP-free) after in vitro infection. We also evaluated the efficacy of the washing procedure recommended by the IETS to decontaminate bovine embryos exposed to C. abortus in vitro. Ninety (8 to 16 cells) bovine embryos, produced in vitro, were randomly divided into 10 batches. Eight batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4.8 × 107 Chlamydia/mL of AB7 strain (ANSES, Maisons-Alfort, France). After incubation for 18 h at 37°C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a PBS and 5% FCS solution without trypsin nor antibiotics in accordance with IETS guidelines. In parallel, 2 batches of 5 embryos (1 ZP-intact and 1 ZP-free) were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13 000 × g. The embryos and wash pellets were tested using RT-PCR. Chlamydia abortus DNA was found in all ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the tenth wash fluid for 1 batch (1/4) of ZP-intact infected embryos and in 3 batches (3/4) of ZP-free infected embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. These results demonstrate that C. abortus adheres to or penetrates the ZP as well as the early embryonic cells of in vitro-produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor cows to healthy recipients or their offspring. Nevertheless, the finding of C. abortus DNA by RT-PCR did not imply that the bacteria found is still infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of bovine embryos infected by C. abortus would eliminate the bacteria from the ZP.

scholarly journals The Effect of pH on Antibiotic Efficacy against Coxiella burnetii in Axenic Media

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cody B. Smith ◽  
Charles Evavold ◽  
Gilbert J. Kersh
Keyword(s):  
Coxiella Burnetii ◽  
Antimicrobial Agents ◽  
Q Fever ◽  
Antimicrobial Treatment ◽  
Etiologic Agent ◽  
Growth And Survival ◽  
Intracellular Compartments ◽  
The Impact

AbstractCoxiella burnetii, the etiologic agent of Q fever, replicates in an intracellular phagolysosome with pH between 4 and 5. The impact of this low pH environment on antimicrobial treatment is not well understood. An in vitro system for testing antibiotic susceptibility of C. burnetii in axenic media was set up to evaluate the impact of pH on C. burnetii growth and survival in the presence and absence of antimicrobial agents. The data show that C. burnetii does not grow in axenic media at pH 6.0 or higher, but the organisms remain viable. At pH of 4.75, 5.25, and 5.75 moxifloxacin, doxycycline, and rifampin are effective at preventing growth of C. burnetii in axenic media, with moxifloxacin and doxycycline being bacteriostatic and rifampin having bactericidal activity. The efficacy of doxycycline and moxifloxacin improved at higher pH, whereas rifampin activity was pH independent. Hydroxychloroquine is thought to inhibit growth of C. burnetii in vivo by raising the pH of typically acidic intracellular compartments. It had no direct bactericidal or bacteriostatic activity on C. burnetii in axenic media, suggesting that raising pH of acidic intracellular compartments is its primary mechanism of action in vivo. The data suggest that doxycycline and hydroxychloroquine are primarily independent bacteriostatic agents.

154 THE QUANTITY OF BOVINE VIRAL DIARRHEA VIRUS ASSOCIATED WITH SINGLE ZONA PELLUCIDA-INTACT IN VITRO-PRODUCED BOVINE EMBRYOS FOLLOWING ARTIFICIAL EXPOSURE

2008 ◽  
Vol 20 (1) ◽  
pp. 157
Author(s):  
J. A. Gard ◽  
M. D. Givens ◽  
P. K. Galik ◽  
K. P. Riddell ◽  
M. S. D. Marley ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Bovine Viral Diarrhea Virus ◽  
Zona Pellucida ◽  
Quantitative Polymerase Chain Reaction ◽  
Bovine Viral Diarrhea ◽  
Bovine Embryos ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

The primary objective of this study was to determine the percentage of individual, preimplantation, in vitro-produced bovine embryos which maintained association with virus despite washing following artificial exposure to a high affinity strain of bovine viral diarrhea virus (BVDV). Another objective of this study was to determine the quantity of virus associated with these embryos. A total of eighty-seven zona pellucida-intact, Day 7, in vitro-produced bovine embryos were exposed for 1 h to 2 � 106 cell culture infected doses per mL to the 50 percent endpoint (CCID50 mL–1) of a type 1 noncytopathic strain of BVDV (SD-1). Following exposure, the embryos were washed according to International Embryo Transfer Society standards for in vitro-produced bovine embryos; they then underwent sonication, RNA extraction, and freezing at –80�C until assayed for virus. A real-time quantitative polymerase chain reaction (QPCR) was run in duplicate on each of the 87 embryos. Forty-two percent (39/87) of the embryos assayed were determined to be positive for virus. The quantity of virus associated with the embryos averaged 0.55 viral copies per 5 µL (SD = 0.89 copies/5 µL, SEM = 0.14 copies/5 µL). Assessment of data using tolerance intervals (P = 0.05) indicates that 90% of contaminated embryos were associated with ≤2.40 viral copies per 5 µL while 99% of contaminated embryos were associated with ≤3.44 viral copies per 5 µL. These findings show that there is a low level of virus associated with in vitro-produced embryos but virus is associated with a significant number of exposed embryos. In conclusion, this study indicates that the potential for transmission of BVDV via embryo transfer of in vitro-produced embryos is small given the amount of virus that was found to associate with individual embryos.

scholarly journals Bacteriostatic and Bactericidal Activities of Tigecycline against Coxiella burnetii and Comparison with Those of Six Other Antibiotics

2009 ◽  
Vol 53 (6) ◽  
pp. 2690-2692 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Anna Psaroulaki ◽  
Iosif Vranakis ◽  
Yannis Tselentis ◽  
Achilleas Gikas
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Vero Cells ◽  
In Vitro Susceptibility ◽  
Shell Vial ◽  
Bactericidal Activities ◽  
Acute Q Fever ◽  
Reference Strains ◽  
Shell Vial Assay

ABSTRACT The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 μg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 μg/ml; linezolid and clarithromycin ranged from 2 to 4 μg/ml; and ciprofloxacin ranged from 4 to 8 μg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 μg/ml.

scholarly journals Coxiella burnetii Interaction with Neutrophils and MacrophagesIn Vitroand in SCID Mice following Aerosol Infection

2013 ◽  
Vol 81 (12) ◽  
pp. 4604-4614 ◽  
Author(s):  
Alexandra Elliott ◽  
Ying Peng ◽  
Guoquan Zhang
Keyword(s):  
Immune Response ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Natural Infection ◽  
Scid Mice ◽  
Innate Immune ◽  
Content Type ◽  
Aerosol Infection

ABSTRACTCoxiella burnetiiis an obligate intracellular bacterium that causes acute and chronic Q fever in humans. Human Q fever is mainly transmitted by aerosol infection. However, there is a fundamental gap in the knowledge regarding the mechanisms of pulmonary immunity againstC. burnetiiinfection. This study focused on understanding the interaction betweenC. burnetiiand innate immune cellsin vitroandin vivo. Both virulentC. burnetiiNine Mile phase I (NMI) and avirulent Nine Mile phase II (NMII) were able to infect neutrophils, while the infection rates were lower than 29%, suggesting thatC. burnetiican infect neutrophils, but infection is limited. Interestingly,C. burnetiiinside neutrophils can infect and replicate within macrophages, suggesting that neutrophils cannot killC. burnetiiandC. burnetiimay be using infection of neutrophils as an evasive strategy to infect macrophages. To elucidate the mechanisms of the innate immune response toC. burnetiinatural infection, SCID mice were exposed to aerosolizedC. burnetii. Surprisingly, neutrophil influx into the lungs was delayed until day 7 postinfection in both NMI- and NMII-infected mice. This result suggests that neutrophils may play a unique role in the early immune response against aerosolizedC. burnetii. Studying the interaction betweenC. burnetiiand the innate immune system can provide a model system for understanding how the bacteria evade early immune responses to cause infection.

scholarly journals Serological survey of antibodies to Toxoplasma gondii and Coxiella burnetii in rodents in north-western African islands (Canary Islands and Cape Verde)

2015 ◽  
Vol 82 (1) ◽  
Author(s):  
Pilar Foronda ◽  
Josué Plata-Luis ◽  
Borja Del Castillo-Figueruelo ◽  
Ángela Fernández-Álvarez ◽  
Aarón Martín-Alonso ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Coxiella Burnetii ◽  
Canary Islands ◽  
Q Fever ◽  
Rattus Rattus ◽  
Cape Verde ◽  
Farm Animals ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
North Western

Coxiella burnetii and Toxoplasma gondii are intracellular parasites that cause important reproductive disorders in animals and humans worldwide, resulting in high economic losses. The aim of the present study was to analyse the possible role of peridomestic small mammals in the maintenance and transmission of C. burnetii and T. gondii in the north-western African archipelagos of the Canary Islands and Cape Verde, where these species are commonly found affecting humans and farm animals. Between 2009 and 2013, 108 black rats (Rattus rattus) and 77 mice (Mus musculus) were analysed for the presence of Coxiella and Toxoplasma antibodies by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IFA), respectively. Our results showed a wide distribution of C. burnetii and T. gondii, except for T. gondii in Cape Verde, in both rodent species. The overall seroprevalence of C. burnetii antibodies was 12.4%; 21.1% for Cape Verde and 10.2% for the Canary Islands. With respect to T. gondii, seropositive rodents were only observed in the Canary Islands, with an overall seroprevalence of 15%. Considering the fact that both pathogens can infect a large range of hosts, including livestock and humans, the results are of public health and veterinary importance and could be used by governmental entities to manage risk factors and to prevent future cases of Q fever and toxoplasmosis.

scholarly journals Development of anEx VivoTissue Platform To Study the Human Lung Response to Coxiella burnetii

2016 ◽  
Vol 84 (5) ◽  
pp. 1438-1445 ◽  
Author(s):  
Joseph G. Graham ◽  
Caylin G. Winchell ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Coxiella Burnetii ◽  
Lung Tissue ◽  
Human Lung ◽  
Q Fever ◽  
Ex Vivo ◽  
Parasitophorous Vacuole ◽  
Inflammasome Activation ◽  
Human Lung Tissue ◽  
Content Type

Coxiella burnetiiis an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells,C. burnetiigenerates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes.C. burnetiirequires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand howC. burnetiievades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infectionin vitroand found that avirulentC. burnetiitriggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection ofex vivohuman lung tissue, defining a valuable approach for characterizingC. burnetiiinteractions with a human host. Within whole lung tissue,C. burnetiipreferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur followingC. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response toC. burnetii.

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