Characteristics of Chlamydia suis Ocular Infection in Pigs

Chlamydia (C.) suis can often be isolated from conjunctival swab specimens from pigs with conjunctivitis or keratoconjunctivitis. In the field, it is assumed to be a multifactorial disease triggered by immunosuppressing factors. This is the first experimental study to provoke clinical signs of conjunctivitis in pigs after C. suis primary mono-infection. Five six-week-old male piglets, free of ocular chlamydia shedding and seronegative for Chlamydia, were conjunctivally infected with the C. suis-type strain S45 (1 × 109 inclusion forming units), while four piglets served as negative controls. The infection group developed clinical signs of conjunctivitis with a peak in the first week post-infection. Immunohistochemical evaluation revealed the presence of Chlamydia not only in the conjunctival epithelium, but also in the enlarged lacrimal glands, lungs, and intestine. No circulating antibodies could be detected during the whole study period of three weeks, although three different test systems were applied as follows: the complement fixation test, MOMP-based Chlamydiaceae ELISA, and PmpC-based C. suis ELISA. Meanwhile, high numbers of IFN-γ-producing lymphocytes within PBMC were seen after C. suis re-stimulation 14 days post-infection. Hence, these data suggest that entry via the eye may not elicit immunological responses comparable to other routes of chlamydial infections.

Transferrins Reduce Replication of Chlamydia suis in McCoy Cells

Chlamydia suis (C. suis) resides in the intestines of pigs and tetracycline-resistant strains are emerging worldwide. Intestinal infections are often subclinical. However, the gut is regarded as a C. suis reservoir and clinical infections have been associated with enteritis, conjunctivitis, pneumonia and reproductive failure. C. suis was found in boar semen and venereal transmission occurred. We studied the anti-Chlamydia suis activity of ovotransferrin (ovoTF) and bovine lactoferrin (bLF). Pre-incubation of C. suis with bLF or ovoTF had no significant effect on overall chlamydia replication (mean fluorescence area) in McCoy cells. The addition of ovoTF to the culture medium had no effect on bacterial replication, but the addition of 0.5 or 5 mg/mL of bLF significantly reduced the inclusion size by 17% and 15% respectively. Egg components are used for cryopreservation of boar semen. When inoculating an ovoTF-containing and Chlamydia suis-spiked semen sample in McCoy cells, a significant reduction in inclusion number (by 7%) and overall replication (by 11%) was observed. Thus, we showed that transferrins possess anti-chlamydial activity. Moreover, ovoTF addition to semen extenders might reduce C. suis venereal transmission. Further research is needed to unravel the mechanisms behind the observations and to enhance the effect of transferrins on C. suis.

Peptide ELISA and FRET-qPCR Identified a Significantly Higher Prevalence of Chlamydia suis in Domestic Pigs Than in Feral Swine from the State of Alabama, USA

Chlamydia suis is an important, highly prevalent, and diverse obligate intracellular pathogen infecting pigs. In order to investigate the prevalence and diversity of C. suis in the U.S., 276 whole blood samples from feral swine were collected as well as 109 fecal swabs and 60 whole blood samples from domestic pigs. C. suis-specific peptide ELISA identified anti-C. suis antibodies in 13.0% of the blood of feral swine (26/276) and 80.0% of the domestic pigs (48/60). FRET-qPCR and DNA sequencing found C. suis DNA in 99.1% of the fecal swabs (108/109) and 21.7% of the whole blood (13/60) of the domestic pigs, but not in any of the assayed blood samples (0/267) in feral swine. Phylogenetic comparison of partial C. suis ompA gene sequences and C. suis-specific multilocus sequencing typing (MLST) revealed significant genetic diversity of the C. suis identified in this study. Highly genetically diverse C. suis strains are prevalent in domestic pigs in the USA. As crowding strongly enhances the frequency and intensity of highly prevalent Chlamydia infections in animals, less population density in feral swine than in domestic pigs may explain the significantly lower C. suis prevalence in feral swine. A future study is warranted to obtain C. suis DNA from feral swine to perform genetic diversity of C. suis between commercial and feral pigs.

Chlamydia suis is associated with intestinal NF-κB activation in experimentally infected gnotobiotic piglets

ABSTRACT Chlamydia suis intestinal infection of single-animal experimental groups of gnotobiotic newborn piglets was previously reported to cause severe, temporary small intestinal epithelium damage. We investigated archived intestinal samples for pro-inflammatory nuclear factor kappa B (NF-κB) activation, Interleukin (IL)-6 and IL-8 production and immune cell influx. Samples were collected 2, 4 and 7 days post-inoculation with C. suis strain S45/6 or mock inoculum (control). Increased nuclear localization of epithelial NF-κB, representative of activation, in the jejunum and ileum of C. suis-infected animals, compared to uninfected controls, began by 2 days post-infection (dpi) and persisted through 7 dpi. Infected animals showed increased production of IL-8, peaking at 2 dpi, compared to controls. Infection-mediated CD45-positive immune cell influx into the jejunal lamina propria peaked at 7 dpi, when epithelial damage was largely resolved. Activation of NF-κB appears to be a key early event in the innate response of the unprimed porcine immune system challenged with C. suis. This results in an acute phase, coinciding with the most severe clinical symptoms, diarrhea and weight loss. Immune cells recruited shortly after infection remain present in the lamina propria during the recovery phase, which is characterized by reduced chlamydial shedding and restored intestinal epithelium integrity.

Comprehensive genome analysis and comparisons of the swine pathogen, Chlamydia suis reveals unique ORFs and candidate host-specificity factors

Chlamydia suis, a ubiquitous swine pathogen, has the potential for zoonotic transmission to humans and often encodes for resistance to the primary treatment antibiotic, tetracycline. Because of this emerging threat, comparative genomics for swine isolate R19 with inter- and intra-species genomes was performed. A 1.094Mb genome was determined through de novo assembly of Illumina high throughput sequencing reads. Annotation and subsystem analyses were conducted, revealing 986 putative genes (Chls_###) that are predominantly orthologs to other known Chlamydia genes. Subsequent comparative genomics revealed a high level of genomic synteny and overall sequence identity with other Chlamydia while 92 unique C. suis open reading frames were annotated. Direct comparison of Chlamydia-specific gene families that included the plasticity zone, inclusion membrane proteins, polymorphic membrane proteins, and the major outer membrane protein, demonstrated high gene content identity with C. trachomatis and C. muridarum. These comparisons also identified diverse components that potentially could contribute to host-specificity. This study constitutes the first genome-wide comparative analysis for C. suis, generating a fully annotated reference genome. These studies will enable focused efforts on factors that provide key species specificity and adaptation to cognate hosts that are attributed to chlamydial infections, including humans.

Mucosal Vaccination with UV-Inactivated Chlamydia suis in Pre-Exposed Outbred Pigs Decreases Pathogen Load and Induces CD4 T-Cell Maturation into IFN-γ+ Effector Memory Cells

Chlamydia trachomatis (Ct) infections are the most frequent bacterial sexually transmitted disease, and they can lead to ectopic pregnancy and infertility. Despite these detrimental long-term sequelae, a vaccine is not available. Success in preclinical animal studies is essential for vaccines to move to human clinical trials. Pigs are the natural host to Chlamydia suis (Cs)—a chlamydia species closely related to Ct, and are susceptible to Ct, making them a valuable animal model for Ct vaccine development. Before making it onto market, Ct vaccine candidates must show efficacy in a high-risk human population. The high prevalence of human Ct infection combined with the fact that natural infection does not result in sterilizing immunity, results in people at risk likely having been pre-exposed, and thus having some level of underlying non-protective immunity. Like human Ct, Cs is highly prevalent in outbred pigs. Therefore, the goal of this study was to model a trial in pre-exposed humans, and to determine the immunogenicity and efficacy of intranasal Cs vaccination in pre-exposed outbred pigs. The vaccine candidates consisted of UV-inactivated Cs particles in the presence or absence of an adjuvant (TriAdj). In this study, both groups of vaccinated pigs had a lower Cs burden compared to the non-vaccinated group; especially the TriAdj group induced the differentiation of CD4+ cells into tissue-trafficking CCR7- IFN-γ-producing effector memory T cells. These results indicate that Cs vaccination of pre-exposed pigs effectively boosts a non-protective immune response induced by natural infection; moreover, they suggest that a similar approach could be applied to human vaccine trials.

Chlamydiales order bacteria in swine with reproductive disoders. Detection of Waddlia chondrophila in swine

Chlamydial infections cause significant economic expenses due to infertility, insufficient productivity growth, and high mortality rates among young stock. Bacteria of the Chlamydiaceae family (Chlamydia suis Chlamydia abortus, Chlamydia psittaci and Chlamydia pecorum) are well known pathogens isolated from pigs. The diseases associated with these species are widely studied; monitoring and prevention are carried out. However, chlamydia-related bacteria in swine remain poorly studied in Ukraine. The purpose of the study was to check samples, taken from swine from private farms in the Poltava region, Ukraine, for the presence of Chlamydia-like organisms. Determination of the possibility of coinfection of Chlamydia and Chlamydia-like organisms. We examined 28 samples from two private farms in the Poltava region (23 samples from sows, and 5 samples from boars). The studies were carried out using PCR with primers designed in our laboratory. As a result of the study, Chlamydiaceae-positive samples were not detected among sows. Along with it, 2 out of 5 samples taken from boars were found to be Chlamydiaceae-positive. The total number of Chlamydiaceae-positive samples was 2 out of 28 (7.14%). The total number of Waddlia-positive samples was 8 out of 28 (28.7%). A mixture of Chlamydiaceae and Waddlia chondrophila infection was found in one sample. Parachlamydia acanthamoebae was not found in any sample. There are no reports of the detection of Waddlia chondrophila in samples from swine in the sources available for us, so this publication might be the first such report. Due to the fact that W. chondrophila was first detected in the samples selected from pigs, the experiment was repeated three times, and the identity of the PCR product of W. chondrophila-positive samples was additionally verified by restriction analysis. Relatively small number of samples, in our research as well as in previous studies, does not allow us to make accurate calculations about the prevalence of chlamydia-like organisms in swine. Further research are required and might include broader sampling, isolation of the pathogen, sequencing of its genome, experimental infection, determination of pathomorphological changes. All the listed above, would help to determine significance of the study along with the role of W. chondrophila in chlamydia infection in swine.

Comparative genomics of swine pathogen Chlamydia suis with human- and mouse-adapted Chlamydia spp

Background The phylum, Chlamydiae, contains a unique set of obligately intracellular bacteria with specific host ranges, despite high genomic relatedness between species. Chlamydia suis, a ubiquitous swine pathogen, has the potential for zoonotic transmission to humans and often encodes for resistance to the primary treatment antibiotic, tetracycline. Because of this emerging threat, and to gain a better understanding of the basic biology, comparative genomics for a tetracycline-resistant swine isolate (R19) with inter- and intra-species genomes was performed. Results A 1.094Mb genome was determined through de novo assembly of Illumina high throughput sequencing reads. Annotation and subsystem analyses were conducted using novel bioinformatic platform, PATRIC, revealing 986 putative genes (Chls_###) that are predominantly orthologs to other known Chlamydia genes. Subsequent comparative genomics using C. trachomatis and C. muridarum, human and mouse pathogens, respectively, revealed a high level of genomic synteny and overall sequence identity. Around 857 genes were found to be conserved between these three species while 92 unique C. suis components were annotated. Direct comparison of chlamydia-specific gene families; inclusion membrane proteins, polymorphic membrane proteins and the major outer membrane protein as well as the plasticity zone, demonstrates the high gene content identity with C. trachomatis and C. muridarum as well as highlights putative host-specificity factors. Conclusions This study constitutes the first genome-wide comparative analysis for C. suis, generating a fully annotated reference genome using NIAID bioinformatic tool, PATRIC. Overall, these analyses build upon previous C. suis analyses to compare the composition of key genetic components with two closely related Chlamydia species. These studies will enable focused efforts on factors that provide key species specificity and adaptation to cognate hosts that are attributed to chlamydial infections, including humans. Continued analysis of the C. suis genome will facilitate studies into the complexities of the chlamydial proteome and discovery of basic biology.

Оновлення таксономічної класифікації мікроорганізмів порядку Chlamydiales

Мета статті – надати актуальну на сьогоднішній день таксономічну класифікацію порядку Chlamydiales та детально роз’яснити терміни: «Chlamydia-related bacteria (CRB)» (хламідіє-споріднені бактерії,) «Chlamydia-like organisms (CLO)» (хламідіє-подібні організми), «environmental chlamydiae» (екологічні хламідії, хламідії довкілля). Методика дослідження. Використано такі методи дослідження: системний аналіз доступних наукових джерел, історичний метод (для вивчення виникнення, формування та розвитку таксономії хламідійних видів у хронологічній послідовності), емпіричний метод (щодо комплексної оцінки сучасного стану об’єкта дослідження), абстрактно-логічний ‒ для уточнення суті основних понять та графічне відображення даних. Результати дослідження. Внаслідок розвитку молекулярної біології за останні 15 років класифікація хламідій зазнала істотних змін і має тенденцію до подальшого вдосконалення. У статті узагальнено історичний досвід і найбільш важливі зміни в таксономії порядку Chlamydiales за останні роки. Ускладнення сучасної класифікації бактерій порядку Chlamydiales свідчить про накопичені знання щодо нових представників цієї групи мікроорганізмів. На сьогодні до порядку Chlamydiales входять 9 родин: Chlamydiaceae, Waddliaceae, Parachlamydiaceae, Criblamydiaceae, Simkaniaceae, Candidatus Clavochlamydiaceae, Candidatus Rhabdochlamydiaceae, Candidatus Piscichlamydia, Candidatus Parilichlamydiaceae, чотири з яких перебувають у статусі кандидатів. До родини Chlamydiaceae, роду Chlamydia тепер належать 14 видів бактерій (Chlamydia abortus, Chlamydia avium, Chlamydia caviae, Chlamydia felis, Chlamydia gallinacea, Chlamydia muridarum, Chlamydia pecorum, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia suis, Chlamydia trachomatis, Candidatus Chlamydia ibidis, Candidatus Chlamydia sanzinia, Candidatus Chlamydia corallus), три з яких перебувають у статусі кандидатів. Родини: Waddliaceae, Parachlamydiaceae, Criblamydiaceae, Simkaniaceae, Ca. Clavochlamydiaceae, Ca. Rhabdochlamydiaceae, Ca. Piscichlamydia, Ca. Parilichlamydiaceae з їхніми численними представниками називають хламідіє-спорідненими бактеріями, або хламідіє-подібними організмами, через їх генетичну і фенотипову подібність та філогенетичну відокремленість від родини Chlamydiaceae. Елементи наукової новизни. У цій статті представлено актуальну таксономічну класифікацію порядку Chlamydiales та надано детально роз’яснено терміни: «Chlamydia-related bacteria (CRB)» (хламідіє-спорідненені бактерії,) «Chlamydia-like organisms (CLO)» (хламідіє-подібні організми), «environmental chlamydiae» (екологічні хламідії, хламідії довкілля). Практична значущість. Надана інформація може бути використана в наукових дослідженнях, впроваджена в навчальний процес при підготовці спеціалістів у галузі ветеринарної медицини, а також використовуватися практикуючими лікарями ветеринарної медицини задля покращення лікування та профілактики хламідіозів тварин та людини. The purpose of the article is to provide the currently relevant taxonomic classification of the Order Chlamydiales, and give a detailed explanation of the terms: “Chlamydia-related bacteria (CRB)”, “Chlamydia-like organisms (CLO)”, "Environmental chlamydiae" (ecological chlamidiae). Research methods. The following research methods were used: system analysis of the available scientific sources, historical method (to study the origin, formation and development of the taxonomy of chlamydia species in chronological order), empirical method (concerning the comlex assessment of the current state of the research object), abstract-logical – to clarify the essence of the basic notions and graphical reflection of the data. The results of the study. As a result of molecular biology development during the recent 15 years, the classification of chlamydia has undergone significant changes and has the tendency to further improvement. This review article is aimed at summarizing the historical experience and the most important changes in the taxonomy of the Order Chlamydiales in recent years. The complication of the modern classification of the Order Chlamydiales bacteria is the inevitable consequence of accumulating knowledge about the new representatives of this group of microorganisms. Consequently at present there are 9 families in the Order Chlamydiales: Chlamydiaceae, Waddliaceae, Parachlamydiaceae, Criblamydiaceae, Simkaniaceae, Candidatus Clavochlamydiaceae, Candidatus Rhabdochlamydiaceae, Candidatus Piscichlamydia, Candidatus Parilichlamydiaceae, four of them are in the status of candidates. Currently, 14 species of bacteria belong to the family Chlamydiaceae, the genus Chlamydia (Chlamydia abortus, Chlamydia avium, Chlamydia caviae, Chlamydia felis, Chlamydia gallinacea, Chlamydia muridarum, Chlamydia pecorum, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia suis, Chlamydia trachomatis, Candidatus Chlamydia ibidis, Candidatus Chlamydia sanzinia, Candidatus Chlamydia corallus), three of which are the candidates. Such families as: Waddliaceae, Parachlamydiaceae, Criblamydiaceae, Simkaniaceae, Ca. Clavochlamydiaceae, Ca. Rhabdochlamydiaceae, Ca. Piscichlamydia, Ca. Parilichlamydiaceae with their numerous representatives are called Сhlamydia-related bacteria, or Сhlamydia-like organisms, due to their genetic and phenotypic similarity, and phylogenetic isolation from the Chlamydiaceae family. The elements of scientific novelty. The currently relevant taxonomic classification of Chlamydiales is presented in the article and also given a detailed explanation of the terms: “Chlamydia-related bacteria (CRB)”, “Chlamydia-like organisms (CLO)”, "Environmental chlamydiae". Practical significance. The provided information can be used in scientific research, introduced into the educational process for training specialists of veterinary medicine, and also be used by practicing veterinary doctors in order to improve the treatment and prevention of animals and human chlamydioses.