Avian chlamydiosis

B

Genotipovi bakterije Chlamydia psittaci u golubova u Hrvatskoj.

In this investigation fecal samples from a total of 232 homing pigeon lofts, belonging to both racing and breeding pigeons, were examined by specific real-time PCR to reveal the presence of Chlamydiaceae, and randomly collected Chlamydia positive samples were genotyped by two different molecular methods; C. psittaci-specific restriction fragment length polymorphism (RFLP) and Multiple-Locus Variable number tandem repeat Analysis (MLVA), in order to obtain relevant information about the prevalence of different genotypes, and to reveal the potential threat to humans that come into close contact with homing pigeons. Chlamydiosis in birds manifests as an acute, unapparent, sub-clinical, and chronic disease, but frequently also as an asymptomatic infection, which represents an additional danger for public health. Currently, nine different genotypes of C. psittaci have been generally accepted, based on PCR amplification, as well as an additional six provisional genotypes, based on DNA microarray. The predominant serotype/genotype in pigeons is B, but also other genotypes were discovered, such as A, C and D, as well as mixed infection. Out of 232 examined samples, 30 (12.9%) of them were PCR positive. DNA from six random positive fecal samples was further examined and the presence of C. psittaci was confirmed in all of them. According to RFLP genotyping, from 6 C. psittaci positive samples, 4 of them belonged to genotype B, and 2 strains remained untyped, due to the low concentration of DNA isolated. Regarding the MLVA typing, the pattern assigned as 22146334 could be described as a classical “pigeon” MLVA pattern. The presence of 12.9% homing pigeons positive to Chlamydiaceae by PCR clearly demonstrates the presence of zoonotic microorganisms and a possible risk for pigeon fanciers that come in close contact with the birds.Ukupno su bila pretražena 232 uzorka izmeta golubova na prisutnost bakterija porodice Chlamydiaceae pomoću za Chlamydiaceae specifične lančane reakcije polimerazom u stvarnom vremenu. Nasumično odabrani pozitivni uzorci pretraženi su i na prisutnost bakterije Chlamydia psittaci (C. psittaci), te su u slučaju pozitivnog nalaza i genotipizirani pomoću dvije različite molekularne metode - MLVA i RFLP, kako bi dobili svrsishodne informacije o učestalosti različitih genotipova, te otkrili potencijalnu prijetnju za zdravlje ljudi koji dolaze u bliži kontakt s golubovima. Klamidioza u ptica očituje se kao akutna, inaparentna, supklinička ili kronična bolest, ali često i kao asimptomatska zaraza, što predstavlja dodatnu opasnost za javno zdravstvo. Novijim istraživanjima, temeljenim na lančanoj reakciji polimerazom (PCR) nađeno je devet različitih genotipova C. psittaci. Prevladavajući serotip/genotip u golubova je B, ali su u njih nađeni i drugi genotipovi, kao što su A, C i D, te mješovite infekcije. Od 232 pregledana uzorka, njih 30 (12,9%) bilo je pozitivno na prisutnost bakterija iz porodice Chlamydiaceae. Nasumično izabrana DNK šest pozitivnih uzoraka dodatno je pretražena i na prisutnost C. psittaci, te je nalaz bio pozitivan u svih šest uzoraka. Pomoću PCR-RFLP metode, od 6 uzoraka pozitivnih na bakteriju C. psittaci, četiri su pripadala genotipu B, dok se dva uzorka nisu mogla pretražiti zbog niske koncentracije izdvojene DNK. Pomoću MLVA metode, četiri pretražena uzorka označena su oznakom 22146334, što se opisuje kao “golublji” genotip. Nalaz 12,9% golubova pozitivnih na prisutnost bakterija porodice Chlamydiaceae jasno pokazuje prisutnost uzročnika u jatima golubova u Hrvatskoj, te mogući rizik za uzgajivače koji dolaze u bliski kontakt s pticama

La psittacose en milieu professionnel avicole

La psittacose est due à une bactérie de petite taille : Chlamydia psittaci. Chez l’homme, elle intervient de façon sporadique ou par foyers groupés. Elle est le plus souvent bénigne et se traduit par des symptômes qui rappellent la grippe. Mais celle-ci peut s’aggraver sous la forme d’une pneumopathie atypique sévère, qui peut être mortelle si un traitement antibiotique n’est pas rapidement mis en place. Le projet "AIRCHLAM" visait à caractériser l’exposition à C. psittaci en milieu professionnel

Genotipovi bakterije Chlamydia psittaci u golubova u Hrvatskoj.

In this investigation fecal samples from a total of 232 homing pigeon lofts, belonging to both racing and breeding pigeons, were examined by specific real-time PCR to reveal the presence of Chlamydiaceae, and randomly collected Chlamydia positive samples were genotyped by two different molecular methods; C. psittaci-specific restriction fragment length polymorphism (RFLP) and Multiple-Locus Variable number tandem repeat Analysis (MLVA), in order to obtain relevant information about the prevalence of different genotypes, and to reveal the potential threat to humans that come into close contact with homing pigeons. Chlamydiosis in birds manifests as an acute, unapparent, sub-clinical, and chronic disease, but frequently also as an asymptomatic infection, which represents an additional danger for public health. Currently, nine different genotypes of C. psittaci have been generally accepted, based on PCR amplification, as well as an additional six provisional genotypes, based on DNA microarray. The predominant serotype/genotype in pigeons is B, but also other genotypes were discovered, such as A, C and D, as well as mixed infection. Out of 232 examined samples, 30 (12.9%) of them were PCR positive. DNA from six random positive fecal samples was further examined and the presence of C. psittaci was confirmed in all of them. According to RFLP genotyping, from 6 C. psittaci positive samples, 4 of them belonged to genotype B, and 2 strains remained untyped, due to the low concentration of DNA isolated. Regarding the MLVA typing, the pattern assigned as 22146334 could be described as a classical “pigeon” MLVA pattern. The presence of 12.9% homing pigeons positive to Chlamydiaceae by PCR clearly demonstrates the presence of zoonotic microorganisms and a possible risk for pigeon fanciers that come in close contact with the birds.Ukupno su bila pretražena 232 uzorka izmeta golubova na prisutnost bakterija porodice Chlamydiaceae pomoću za Chlamydiaceae specifične lančane reakcije polimerazom u stvarnom vremenu. Nasumično odabrani pozitivni uzorci pretraženi su i na prisutnost bakterije Chlamydia psittaci (C. psittaci), te su u slučaju pozitivnog nalaza i genotipizirani pomoću dvije različite molekularne metode - MLVA i RFLP, kako bi dobili svrsishodne informacije o učestalosti različitih genotipova, te otkrili potencijalnu prijetnju za zdravlje ljudi koji dolaze u bliži kontakt s golubovima. Klamidioza u ptica očituje se kao akutna, inaparentna, supklinička ili kronična bolest, ali često i kao asimptomatska zaraza, što predstavlja dodatnu opasnost za javno zdravstvo. Novijim istraživanjima, temeljenim na lančanoj reakciji polimerazom (PCR) nađeno je devet različitih genotipova C. psittaci. Prevladavajući serotip/genotip u golubova je B, ali su u njih nađeni i drugi genotipovi, kao što su A, C i D, te mješovite infekcije. Od 232 pregledana uzorka, njih 30 (12,9%) bilo je pozitivno na prisutnost bakterija iz porodice Chlamydiaceae. Nasumično izabrana DNK šest pozitivnih uzoraka dodatno je pretražena i na prisutnost C. psittaci, te je nalaz bio pozitivan u svih šest uzoraka. Pomoću PCR-RFLP metode, od 6 uzoraka pozitivnih na bakteriju C. psittaci, četiri su pripadala genotipu B, dok se dva uzorka nisu mogla pretražiti zbog niske koncentracije izdvojene DNK. Pomoću MLVA metode, četiri pretražena uzorka označena su oznakom 22146334, što se opisuje kao “golublji” genotip. Nalaz 12,9% golubova pozitivnih na prisutnost bakterija porodice Chlamydiaceae jasno pokazuje prisutnost uzročnika u jatima golubova u Hrvatskoj, te mogući rizik za uzgajivače koji dolaze u bliski kontakt s pticama

Emerging Chlamydia psittaci infections in chickens and examination of transmission to humans

Chlamydia psittaci and atypical Chlamydiaceae infections are (re)-emerging in chickens. We therefore examined the prevalence of C. psittaci, atypical Chlamydiaceae and their zoonotic transmission on 19 Belgian chicken farms. Atypical Chlamydiaceae were not detected in chickens but 18 out of 19 farms were positive for C. psittaci by culture and PCR. C. psittaci ompA genotypes A and D were discovered. None of the examined humans (n = 31) was infected with atypical Chlamydiaceae, but 29 (93.5%) of them were positive for C. psittaci by culture and PCR. Genotypes A, D and a mixed infection with genotypes C and D were found. Humans (n = 2) working at the C. psittaci-negative farm never had respiratory complaints, while 25 out of 29 positive farmers (86.2%) reported yearly medical complaints potentially related to psittacosis. Four of them currently experienced respiratory disease and one of them was being treated with antibiotics. Four farmers (12.5%) mentioned that they had pneumonia after starting to keep chickens. Occupational physicians should be aware of emerging Chlamydiaceae infections in chickens

Identifikacija i MLVA genotipizacija vrste Chlamydia abortus iz slučajeva pobačaja malih preživača u Hrvatskoj

In addition to zoonotic potential, Chlamydia (C.) abortus is a very important bacterium causing serious disease in small ruminants. The main outcome of the disease is abortion in the late stages of pregnancy and the economic impact for farms is significant. During a three-year period (2015–2017), 191 vaginal swabs, 24 placentas, 210 foetal organs and 2 milk samples from small ruminant abortion cases were tested for C. abortus by real-time PCR. Positive samples were detected on eight sheep farms and two goat farms, with 8.4% of total samples testing positive samples. These samples were characterised using the MLVA method, and a single MLVA genotype (genotype [2]) was identified from sheep and goat samples, suggesting highly conserved C. abortus strains among the national flock. This study is the first description of C. abortus as a causative agent of abortion in goats in Croatia. More detailed study is required to recognize the epidemiological relevance of the abortion chlamydiosis. An open register of farms with defined health status should be established for each farm at the national level for better disease(s) control.Osim zoonotskog potencijala, vrsta Chlamydia (C.) abortus je vrlo važna bakterija koja može prouzročiti tešku bolest malih preživača. Dominantni klinički znak bolesti je pobačaj u kasnom stadiju graviditeta sa znatnim ekonomskim učinkom za farme. Tijekom trogodišnjeg razdoblja (2015.-2017.) metodom Real time (RT) PCR na C. abortus testiran je 191 vaginalni obrisak, 24 placente, 210 organa fetusa i 2 uzorka mlijeka iz slučajeva pobačaja malih preživača. Pozitivni uzorci ustvrđeni su u 8 farmi ovaca i 2 farme koza, što ukupno predstavlja 8,4 % pozitivnih uzoraka. Ovi uzorci su tipizirani metodom MLVA i identificiran je jedinstveni genotip MLVA (genotip [2]). Dobiveni rezultat ukazuje na visok stupanj podudarnosti sojeva C. abortus u nacionalnom stadu ovaca i koza. Ovo istraživanje prvi je opis vrste C. abortus kao uzročnika pobačaja u koza u Hrvatskoj. Temeljem ustvrđenog rezultata bilo bi uputno provesti daljnja, detaljnija istraživanja da bi se prepoznalo epidemiološko i ekonomsko značenje infekcija vrstom C. abortus. Radi kvalitetnijeg nadzora bolesti potrebno je uspostaviti otvoreni registar gospodarstava s definiranim zdravstvenim statusom za svaku pojedinu farmu

Multiple-locus variable-number tandem repeat analysis for molecular typing of Aspergillus fumigatus

<p>Abstract</p> <p>Background</p> <p>Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related microbial isolates to provide information for establishing genetic patterns among isolates and to investigate disease outbreaks. The usefulness of MLVA was recently demonstrated for the avian major pathogen <it>Chlamydophila psittaci</it>. In the present study, we developed a similar method for another pathogen of birds: the filamentous fungus <it>Aspergillus fumigatus</it>.</p> <p>Results</p> <p>We selected 10 VNTR markers located on 4 different chromosomes (1, 5, 6 and 8) of <it>A. fumigatus</it>. These markers were tested with 57 unrelated isolates from different hosts or their environment (53 isolates from avian species in France, China or Morocco, 3 isolates from humans collected at CHU Henri Mondor hospital in France and the reference strain CBS 144.89). The Simpson index for individual markers ranged from 0.5771 to 0.8530. A combined loci index calculated with all the markers yielded an index of 0.9994. In a second step, the panel of 10 markers was used in different epidemiological situations and tested on 277 isolates, including 62 isolates from birds in Guangxi province in China, 95 isolates collected in two duck farms in France and 120 environmental isolates from a turkey hatchery in France. A database was created with the results of the present study <url>http://minisatellites.u-psud.fr/MLVAnet/</url>. Three major clusters of isolates were defined by using the graphing algorithm termed Minimum Spanning Tree (MST). The first cluster comprised most of the avian isolates collected in the two duck farms in France, the second cluster comprised most of the avian isolates collected in poultry farms in China and the third one comprised most of the isolates collected in the turkey hatchery in France.</p> <p>Conclusions</p> <p>MLVA displayed excellent discriminatory power. The method showed a good reproducibility. MST analysis revealed an interesting clustering with a clear separation between isolates according to their geographic origin rather than their respective hosts.</p

Genomic evidence that the live Chlamydia abortus vaccine strain 1B is not attenuated and has the potential to cause disease.

BACKGROUND: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis. METHODS: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains). RESULTS: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains. CONCLUSIONS: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease

Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens

Glanders - an almost forgotten zoonosis

Maleus je kontagiozna bolest konja, magaraca i njihovih križanaca, a mogu oboljeti i mesojedi, uključujući i čovjeka. Obično je kronična, a očituje se tvorbom specifičnih čvorića u koži, potkožju, plućima i drugdje u organizmu. U Hrvatskoj je bolest zadnji puta zabilježena 1961. godine. U Arhivi sojeva Laboratorija za bakterijske zoonoze, Hrvatskog veterinarskog instituta pronađen je liofilizat oznake B. mallei - Karlovac (5.2.1957.). Zbog iznimno rijetkog uzorka namjera je bila ponovno kultivirati soj te ga identificirati daljnjim standardnim i molekularnim testovima potvrditi radi li se zaista o vrsti Burkholderia (B.) mallei. Liofilizirani izolat B. mallei nismo uspjeli kultivirati opisanim bakteriološkim postupcima. Izvršili smo izolaciju DNK iz uzorka otopljenog liofilizata. Koncentracija izolirane DNK izmjerena je na DS-11 spektrofotometru (DeNovix, SAD). Umnožavanjem za vrstu B. mallei specifičnog fliP-IS407A odsječka veličine 989-bp za soj „B.mallei Karlovac 5.2.1957.“ 221. utvrđeno je da se radi o vrsti B. mallei. Prema trenutno dostupnim podatcima molekularne genotipizacije primjenom high resolution melting PCR metode na 15 filogenetski značajnih polimorfizama jedne baze vrste B. mallei, hrvatski soj izdvojen 1957. godine nalazi se u grupi filogenetski srodnih sojeva iz Italije, Mađarske, Turske i Irana iz istog vremenskog razdoblja. Maleus je klasična, reemergentna bakterijska zoonoza i bolest koja ne poznaje granice kako po klinički nespecifičnoj slici i kroničnim oblicima bolesti bez vidljivih simptoma tako i po činjenici da se slučajevi bolesti u konja dijagnosticiraju i danas u zemljama s višegodišnjim statusom bez bolesti. Kontrola konja u međunarodnom prometu je i dalje neophodna.Glanders is a contagious disease of horses, donkeys and their crossbreeds, though it is also known to infect carnivores, including humans. The disease is usually chronic and is manifested by the formation of specific nodes in skin, subcutaneous tissue, lungs and elsewhere. In Croatia, the last record of the disease was in 1961. In the archive of strains in the Laboratory for Bacterial Zoonoses at the Croatian Veterinary Institute, a lyophilisate marked B. mallei – Karlovac (5.2.1957) was found. As these specimens are exceptionally rare, the intention was to re-cultivate the strain and identify it with further standard and molecular tests, to confirm whether it was really Burkholderia (B.) mallei. The lyophilized isolate could not be cultivated using the described bacteriological procedures. DNA extraction from the sample of dissolved lyophilizate was performed and a concentration of extracted DNA was measured on the DS-11 Spectrophotometer (DeNovix, USA). By amplifying B. mallei specific, 989- bp long, flip-IS407A region, it was possible to determine that the strain “B. mallei Karlovac 5.2.1957” belongs to the B. mallei species. According to the currently available data on molecular genotyping using high-resolution melting PCR method on 15 phylogenetically significant single nucleotide polymorphisms of B. mallei, the Croatian strain isolated in 1957 falls within the group of phylogenetically related strains from Italy, Hungary, Turkey and Iran from the same time period. Glanders is a classical, re-emerging bacterial zoonosis and a disease that knows no boundaries, both in the clinically unspecific and chronic forms of illness without visible symptoms, and in the fact that the cases of the disease in horses are still diagnosed today in countries with long- term disease-free status. The control of horses in international traffic is still necessary