Antonietta Di Francesco

Selection for tetracycline-resistant Chlamydia suis in treated pigs

The aim of this study was to investigate Chlamydia suis in a pig farm with an outbreak of conjunctivitis and diarrhea. Eye swabs and pooled fecal samples were investigated for the presence of C. suis by real-time PCR and ArrayTube microarray. Samples positive for C. suis by ArrayTube microarray assay were further tested for the presence of the tet(C) resistance gene by PCR. In the first examination, C. suis was identified in 12 six-week-old pigs showing conjunctivitis. Of these, the tet(C) gene-coding region was amplified in one pooled fecal sample and one eye swab, respectively. After oral treatment with tetracycline, clinical symptoms disappeared. Subsequently, all eye swabs investigated from 10 healthy pigs were positive for C. suis and the tet(C) gene-coding region. The present study reports rapid selection for tetracycline-resistant C. suis after antibiotic treatment.

Prevalence of Chlamydial Infections in Fattening Pigs and Their Influencing Factors

Chlamydial infections in pigs are associated with respiratory disease, diarrhea, conjunctivitis and other pathologies. The aim of this study was to define the prevalence of Chlamydiaceae in Swiss fattening pigs by applying sensitive and specific detection methods and to correlate prior antibiotic treatment and farm related factors with differences in prevalence. Conjunctival and fecal swabs were collected from 636 pigs in 29 Swiss fattening pig farms with and without antibiotic treatment, at the beginning and the end of the fattening period. The swabs were screened by real-time PCR for Chlamydiaceae. For the chlamydial detection and species-identification, a DNA-microarray analysis was performed. All farms were positive for Chlamydiaceae with 94.3 and 92.0% prevalence in fecal swabs as well as 45.9 and 32.6% in conjunctival swabs at the first and second time points, respectively. Antibiotic treatment could not clear the infection on herd level. Potential contact with wild boars was a significant risk factor, while hygiene criteria did not influence chlamydial prevalence. A correlation of chlamydial positivity to diarrhea, but not to conjunctivitis was evident. Chlamydia suis was the predominant species. Mixed infections with C. suis and C. pecorum were common, with a substantial increase in C. pecorum positivity at the end of the fattening period, and this finding was associated with ruminant contact. C. abortus was detected in one conjunctival swab. In this study, C. suis inhabited the intestinal tract of nearly all examined pigs, implying a long-term infection. C. pecorum was also common and might be transmitted to pigs by ruminants.

In Vitro Activities of Several Antimicrobial Agents against Recently Isolated and Genotyped Chlamydia trachomatis Urogenital Serovars D through K

ABSTRACT A systematic evaluation of the susceptibility of all Chlamydia trachomatis urogenital serovars (D through K) to levofloxacin, erythromycin, doxycycline, clarithromycin, and azithromycin was performed. All C. trachomatis serovars had comparable susceptibilities with respect to the various antimicrobials tested, thus confirming the homogeneous data so far obtained regarding the susceptibility of C. trachomatis to antimicrobial agents.

Serological response to pgp3 protein in animal and human chlamydial infections.

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.

Seroepidemiologic Survey for Chlamydia suis in Wild Boar (Sus scrofa) Populations in Italy

We used serology to estimate the prevalence of exposure to chlamydiae in Italian populations of wild boars (Sus scrofa). Sera from 173 hunter-killed wild boars harvested during the 2006–2009 hunting seasons in three Italian regions were tested for antibodies to Chlamydia suis, Chlamydophila pecorum, Chlamydophila abortus, and Chlamydophila psittaci by the microimmunofluorescence test. Antibody titers to chlamydiae ≥1:32 were detected in 110 of the 173 samples tested (63.6%). Specific reactivity could be assessed only in 44 sera with antibody titers to C. suis that were two- to threefold higher than antibody titers against the other chlamydial species; the other 66 sera had similar reactivity against all the chlamydia species tested. Antibody to C. suis was detected in sera from wild boar populations with rare or no known contact with domestic pigs. These results suggest that the wild boar could be a chlamydia reservoir and may acquire chlamydiae independent of contacts with the domestic pig.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination.

ABSTRACT Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.

Tetracycline Susceptibility in Chlamydia suis Pig Isolates

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 μg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

Host defense peptides: general overview and an update on their activity against Chlamydia spp.

Chlamydiae are obligate intracellular bacteria that cause serious diseases in a wide range of hosts. Chlamydia trachomatis is one of the leading sexually transmitted pathogens in the world. Because vaccines are not currently available, effective drugs are essential. In both animals and humans, chlamydial infections are often treated with tetracycline or its derivatives. A stable tetracycline-resistant phenotype was described in Chlamydia suis strains from pigs in the USA and in Europe. In humans, there are reports of tetracycline treatment failure and the in vitro adaptability of C. trachomatis to evolve to antibiotic resistance has been described, suggesting the pressing need to search for alternative and effective classes of antimicrobial drugs. Host defense peptides (HDPs) are known as direct antimicrobial agents as well as innate immune modulators. Being active against multidrug-resistant bacteria, HDPs are attractive candidates as templates for new drugs. A number of studies evaluated the activity of natural and synthetic HDPs against Chlamydia spp., showing C. trachomatis to be the most sensitive among chlamydia species tested. Protegrins and α-helical peptides were the most active among the HDPs assessed.

Selective Pressure Promotes Tetracycline Resistance of Chlamydia Suis in Fattening Pigs.

In pigs, Chlamydia suis has been associated with respiratory disease, diarrhea and conjunctivitis, but there is a high rate of inapparent C. suis infection found in the gastrointestinal tract of pigs. Tetracycline resistance in C. suis has been described in the USA, Italy, Switzerland, Belgium, Cyprus and Israel. Tetracyclines are commonly used in pig production due to their broad-spectrum activity and relatively low cost. The aim of this study was to isolate clinical C. suis samples in cell culture and to evaluate their antibiotic susceptibility in vitro under consideration of antibiotic treatment on herd level. Swab samples (n = 158) identified as C. suis originating from 24 farms were further processed for isolation, which was successful in 71% of attempts with a significantly higher success rate from fecal swabs compared to conjunctival swabs. The farms were divided into three treatment groups: A) farms without antibiotic treatment, B) farms with prophylactic oral antibiotic treatment of the whole herd consisting of trimethoprime, sulfadimidin and sulfathiazole (TSS), or C) farms giving herd treatment with chlortetracycline with or without tylosin and sulfadimidin (CTS). 59 isolates and their corresponding clinical samples were selected and tested for the presence or absence of the tetracycline resistance class C gene [tet(C)] by conventional PCR and isolates were further investigated for their antibiotic susceptibility in vitro. The phenotype of the investigated isolates was either classified as tetracycline sensitive (Minimum inhibitory concentration [MIC] < 2 μg/ml), intermediate (2 μg/ml ≤ MIC < 4 μg/ml) or resistant (MIC ≥ 4 μg/ml). Results of groups and individual pigs were correlated with antibiotic treatment and time of sampling (beginning/end of the fattening period). We found clear evidence for selective pressure as absence of antibiotics led to isolation of only tetracycline sensitive or intermediate strains whereas tetracycline treatment resulted in a greater number of tetracycline resistant isolates.

Antibody-neutralizing activity against all urogenital Chlamydia trachomatis serovars in Chlamydia suis-infected pigs

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.