Evidence for decontamination of single-use filtering facepiece respirators.

Single-use filtering face respirators (FFRs) are critical pieces of personal protective equipment for healthcare workers treating patients with suspected upper respiratory tract pathogens. Experiences during pandemics in the 2000s, as well as the ongoing COVID-19 pandemic caused by the SARS-2-CoV-2, have highlighted concerns over the pressures that sustained respiratory virus pandemics may have on supplies of FFRs globally. Decontamination of FFRs has been posited as one solution to support the re-use of FFRs with a growing body of literature over the last 10+ years beginning to examine both the efficacy of disinfection of contaminated FFRs but also the impact of the decontamination process on the FFR's performance. Physical and chemical methods of decontamination have been tested for treatment of FFRs with ultraviolet germicidal irradiation, sterilization by steam, ethylene oxide and vaporous hydrogen peroxide, demonstrating the most promising results thus far. Many of these methods utilize existing equipment that may already be available in hospitals and could be re-purposed for FFR decontamination. Importantly, some methods may also be replicated on household equipment, broadening the utility of FFR decontamination across a range of healthcare settings. Utilizing techniques to experimentally contaminate FFRs with a range of microorganisms, most decontamination methods appear to reduce the risk of the mask as a source of infection to the wearer and others to negligible levels. The performance of the filter, especially the efficiency of particle penetration following treatment, varied greatly depending on the processing method as well as the model of the filter itself, however. Urgent regulatory body-supported research is required to endorse the routine decontamination of FFRs. In emergency settings, these methods should nevertheless be carefully considered as one strategy to address potential shortfalls in supplies of FFRs for healthcare workers.

Chlamydial infection and on-farm risk factors in dairy cattle herds in South East Queensland.

Chlamydial infections in dairy cattle are common and have been sporadically associated with reduced performance and severe disease manifestations. While chlamydial infections are well described in sheep, very little is known about the epidemiology of these infections in dairy cattle in Australia. In this study, we screened for chlamydial infections and assessed on-farm risks in dairy cattle herds from Southeast Queensland (SE Qld) region of Australia. In total, 228 paired vaginal and rectal swabs were collected from 114 visually healthy dairy cows from four farms in SE Qld. Risk factors were rated by observational study and included: hygiene and cleanliness of cows, walkway and parlour, incidence of perinatal mortality, external replacements, mode of breeding, calving pen management, heat reduction strategies, and feed ration usage. Testing for chlamydial pathogens (Chlamydia pecorum, Chlamydia psittaci and Chlamydia abortus) was done using species-specific quantitative polymerase chain reaction (qPCR) assays. Detected rates of chlamydial infection were evaluated against the on-farm risk factors. C. pecorum infection was widespread in all four farms, with 56.1% (64/114) of individual animals shedding this organism from vaginal and rectal, or both sites. C. abortus and C. psittaci were not detected in any animals. No association was found to exist with risk factors and C. pecorum infection rates in our study, however the number of Chlamydia positive animals was statistically different between the herds. This study suggests that subclinical chlamydial infections may impact on dairy herd health at the production level rather than affecting individual animal.

Detection of Chlamydiaceae in ocular swabs from Australian pre-export feedlot sheep.

Infectious Ovine Keratoconjunctivitis (IOK) is a contagious ocular disease of sheep. A range of organisms have been observed as the aetiological agents of IOK. In this study, the presence of chlamydial pathogens (C. pecorum, C. abortus, C. psittaci) in conjunctival swabs was tested for. The swabs were collected from sheep with varying grades of IOK in an Australian pre-export feedlot. The sheep had been rejected from a shipment because of the eye disease. The relative contribution of chlamydial pathogens to IOK and the rejection of animals was evaluated. In total, 149 conjunctival swabs were taken from rejected sheep (IOK Grades 1 to 6; n = 126) as well as those with healthy eyes (Grade 0; n = 23). Screening for chlamydial pathogens was done using species-specific qPCR assays. Chlamydial DNA was detected in 35.6% (53/149) of conjunctival samples. C. pecorum was the most predominant species with an overall prevalence of 28.9% (43/149). C. psittaci prevalence was 6.7% (10/149). Both organisms were detected in healthy as well as IOK-affected eyes. All swabs tested negative for C. abortus. The results from this study demonstrate that Chlamydia spp can be readily detected in sheep presenting with IOK. The zoonotic C. abortus was not detected in any of the samples in this study, providing further evidence to the suggestion that this pathogen remains absent from Australia. Although the exact contribution of Chlamydia spp in the IOK pathogenesis is unclear, such studies are anticipated to be of benefit to Australian domestic and live export production systems.

Molecular evidence to suggest pigeon-type Chlamydia psittaci in association with an equine foal loss.

Chlamydia psittaci is an important avian pathogen with spillover from infected wild and domesticated birds also posing a risk to human health. We recently reported a case of C. psittaci equine placentitis associated with further spillover to humans. Molecular typing of this case revealed it belonged to the 6BC clade of C. psittaci, a globally distributed highly virulent set of strains, typically linked to infection spillover from parrots. Equine chlamydiosis associated with C. psittaci infection has previously been reported elsewhere in countries where parrots are not endemic, however, raising questions over the identity of infecting C. psittaci strains and the potential infection reservoirs. In this study, we describe the detection and molecular characterization of C. psittaci in a case of equine abortion in southern Queensland. Equine placenta and fresh liver and lung tissue from the necropsied foetus were positive by C. psittaci-specific qPCR. Chlamydia psittaci-specific multilocus sequence typing and ompA genotyping were used to further characterize the detected equine strains and an additional strain obtained from a dove from a different geographic region presenting with psittacosis. Molecular typing of this case revealed that the infecting equine strains were closely related to the C0sittaci detected in dove, all belonging to an evolutionary lineage of C. psittaci strains typically associated with infections of pigeons globally. This finding suggests a broader diversity of C. psittaci strains may be detected in horses and in association with reproductive loss, highlighting the need for an expansion of surveillance studies globally to understand the epidemiology of equine chlamydiosis and the associated zoonotic risk.

New and emerging chlamydial infections of creatures great and small.

Until recently, our knowledge of the host range and diversity of members of the Chlamydiaceae, obligate intracellular bacterial pathogens of humans and animals, was thought to be nearly complete. Aided by advances in molecular diagnostics, a new picture is emerging, however, that the host barriers may be looser than previously thought for many chlamydial species. While cross-host transmission of chlamydial species is a concern for animal health, new reports highlight an emerging zoonotic risk for several species associated with intensification of farming and the widespread popularity of companion animals. The description of an expanded cohort of new species within this family from avian and reptilian hosts has also highlighted how much we still have to learn about the biology and pathogenicity of the Chlamydiaceae as a whole. Reports emerging about these relatives of the traditional chlamydial pathogens are matched by the continued identification of novel Chlamydia-related bacteria in the phylum Chlamydiae, providing evidence that many may be pathogenic to humans or animals and pose a zoonotic or vector-borne risk. The review examines the new hosts described for well-characterized chlamydial veterinary pathogens, emerging novel chlamydial species and the potential for these to cause disease in their respective hosts.

Chlamydial infections of fish: diverse pathogens and emerging causes of disease in aquaculture species.

Chlamydial infections of fish are emerging as an important cause of disease in new and established aquaculture industries. To date, epitheliocystis, a skin and gill disease associated with infection by these obligate intracellular pathogens, has been described in over 90 fish species, including hosts from marine and fresh water environments. Aided by advances in molecular detection and typing, recent years have seen an explosion in the description of these epitheliocystis-related chlamydial pathogens of fish, significantly broadening our knowledge of the genetic diversity of the order Chlamydiales. Remarkably, in most cases, it seems that each new piscine host studied has revealed the presence of a phylogenetically unique and novel chlamydial pathogen, providing researchers with a fascinating opportunity to understand the origin, evolution and adaptation of their traditional terrestrial chlamydial relatives. Despite the advances in this area, much still needs to be learnt about the epidemiology of chlamydial infections in fish if these pathogens are to be controlled in farmed environments. The lack of in vitro methods for culturing of chlamydial pathogens of fish is a major hindrance to this field. This review provides an update on our current knowledge of the taxonomy and diversity of chlamydial pathogens of fish, discusses the impact of these infections on the health, and highlights further areas of research required to understand the biology and epidemiology of this important emerging group of fish pathogens of aquaculture species.

Molecular characterization of "Candidatus Similichlamydia latridicola" gen. nov., sp. nov. (Chlamydiales: "Candidatus Parilichlamydiaceae"), a novel Chlamydia-like epitheliocystis agent in the striped trumpeter, Latris lineata (Forster).

Histological analysis of gill samples taken from individuals of Latris lineata reared in aquaculture in Tasmania, Australia, and those sampled from the wild revealed the presence of epitheliocystis-like basophilic inclusions. Subsequent morphological, in situ hybridization, and molecular analyses were performed to confirm the presence of this disease and discovered a Chlamydia-like organism associated with this condition, and the criteria set by Fredericks and Relman's postulates were used to establish disease causation. Three distinct 16S rRNA genotypes were sequenced from 16 fish, and phylogenetic analyses of the nearly full-length 16S rRNA sequences generated for this bacterial agent indicated that they were nearly identical novel members of the order Chlamydiales. This new taxon formed a well-supported clade with "Candidatus Parilichlamydia carangidicola" from the yellowtail kingfish (Seriola lalandi). On the basis of sequence divergence over the 16S rRNA region relative to all other members of the order Chlamydiales, a new genus and species are proposed here for the Chlamydia-like bacterium from L. lineata, i.e., "Candidatus Similichlamydia latridicola" gen. nov., sp. nov.

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

Olfactory neuroblastoma in dogs and cats--a histological and immunohistochemical analysis.

Olfactory neuroblastoma (ONB) was identified in 13 dogs and nine cats. The tumours were subjected to microscopical examination and were graded using a human pathological grading system. In the canine and feline tumours there was more necrosis and higher mitotic activity (mitotic index and Ki67 labelling index) than reported in human ONB. Rosettes were a common feature of feline ONBs. A significant correlation was observed between the histological grade and the Ki67 labelling index. The histopathological diagnosis of ONB was confirmed immunohistochemically by demonstration of the neuronal marker neuron-specific enolase (NSE). Two other neuron-specific antibodies specific for microtubule-associated protein-2 (MAP-2) and neuronal nuclei antigen (NeuN) were evaluated. MAP-2 expression proved to have higher specificity than labelling for NSE. NeuN expression was less sensitive and of limited practical value.

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus).

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.

Novel Chlamydiales associated with epitheliocystis in a leopard shark Triakis semifasciata.

The Chlamydiales is a diverse order of obligate intracellular gram-negative bacteria that are known to cause a wide range of diseases in terrestrial animals, including humans. Molecular analyses have revealed that these organisms are also associated with epitheliocystis in teleost fish species, highlighting the suspected deep evolutionary origin of members of this bacterial order. However, our knowledge of their fish host range and of the diversity of the bacteria themselves is still very limited. In this study, we provide molecular evidence for a novel member of the Order Chlamydiales in a nonteleost species, the leopard shark Triakis semifasciata. Based on phylogenetic analysis of the 16S rRNA gene, this novel organism appears to represent a unique lineage in the Order Chlamydiales despite appearing histologically similar to epitheliocystis-causing organisms in other fish species. A greater understanding of the genetic diversity of marine Chlamydiales will assist our attempts to manage and control epitheliocystis outbreaks and to understand the evolution of this unique obligate intracellular pathogen.

Immunohistochemical detection of survivin in canine lymphoma.

Survivin is a member of the family of proteins known as 'inhibitors of apoptosis proteins'. Survivin has a role in cellular decisions concerning division and survival and is frequently expressed in neoplastic cells. The aim of the present study was to investigate immunohistochemically the expression of survivin in normal canine tissues and in canine lymphoma. A representative range of fetal and adult normal tissues as well as biopsy samples from dogs with lymphoma were assembled in tissue arrays. The lymphomas were classified according to the revised Kiel and to the Revised European American Lymphoma-World Health Organization (REAL-WHO) schemes. Polyclonal and monoclonal antisera cross-reactive with canine survivin identified cytoplasmic expression of the molecule in a broad range of normal canine cells. The same reagents demonstrated cytoplasmic labelling of more than 5% of cells in all 83 lymphoma samples tested with polyclonal antiserum and in 67 of 82 (82%) of samples tested with monoclonal antiserum. Survivin was expressed by a wide range of canine lymphoma subtypes, but the expression of this molecule in normal canine tissues must be considered if novel therapies targeting survivin are applied to the management of canine lymphoma.

Molecular evidence for chlamydial infections in the eyes of sheep.

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.

Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks.

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.

Detection of chlamydiae in boar semen and genital tracts.

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.

Immunohistochemical study about the Flt-1/VEGFR1 expression in the gastrointestinal tract of mouse, rat, dog, swine and monkey.

Fms-like tyrosine kinase 1 (Flt-1) performs a subordinate effector role in mesenchymal angiogenesis and potentially serves an equally important functional role as a self-contained receptor in epithelial cells. In both endothelial cells and epithelial cells, Flt-1/vascular endothelial growth factor receptor 1 (VEGFR1) downstream signalling is involved in regulating cellular processes such as cytoskeletal changes and cellular survival protection. Cellular renewal of the gastrointestinal mucosa is based on these processes and might involve Flt-1/VEGFR1 pathway activities; the molecular mechanisms regulating these cellular dynamics remain unclear. This study was performed to investigate the presence and distribution of Flt-1/VEGFR1 in epithelial cells of the gastrointestinal tract by immunohistochemistry (IHC). Gastrointestinal tissues were taken from eight anatomical sites from mouse, rat, dog, swine and monkey. Present results revealed a cytosolic Flt-1/VEGFR1 staining pattern in mucosal epithelial cells for all investigated species. Non-epithelial structures also displayed a distinct Flt-1/VEGFR1 positivity and included vascular smooth muscle walls, enteric smooth muscle layers, the enteric nervous system and capillary endothelial cells. Diverse intensities of the Flt-1/VEGFR1 binding reaction within each species were observed in the intestinal mucosa with a strong immunoreaction in enterocytes and with a low protein expression in the ileum in most species. Crypt cells in the large intestine were mostly negative for Flt-1/VEGFR1. A peculiar and mainly intranuclear antibody binding reaction was found in Brunner's gland epithelial cells of mouse and rat whereas Brunner's glands of dog, swine and monkey remained completely negative. These results indicate a potential involvement of Flt-1/VEGFR1 in normal restitution of gastrointestinal structures in the species studied. Additionally, intranuclear Flt-1/VEGFR1 antibody binding in Brunner's glands of rodents may suggest a nuclear translocation of the transmembrane VEGFR1 which has not previously been described.

Detection of mycobacteria and chlamydiae in granulomatous inflammation of reptiles: a retrospective study.

A retrospective study on reptile tissues presenting with granulomatous inflammation was performed to detect the possible presence of mycobacteria and chlamydiae in these lesions. Ninety cases including 48 snakes, 27 chelonians, and 15 lizards were selected. Mycobacteria were detected by Ziehl-Neelsen (ZN) staining and a broad-range polymerase chain reaction (PCR) followed by DNA sequencing. To detect chlamydiae, immunohistochemistry with monoclonal antibodies against chlamydial lipopolysaccharide (LPS) and a Chlamydiales order-specific PCR and sequencing were applied. Acid-fast bacilli were found in 14 cases (15.6%) by ZN staining and in 23 cases (25.6%) by PCR. Sequence analysis revealed the presence of Mycobacteria other than Mycobacterium tuberculosis complex (MOTT). Chlamydial LPS antigen was observed within granulomas from five samples (5.6%), whereas the PCR screen revealed 58 positive cases (64.4%). Of these, 9 cases (10%) showed 98-99% similarity to Chlamydophila (Cp.) pneumoniae and 49 cases (54.4%) displayed a high similarity (88-97%) to the newly described "Chlamydia-like" microorganisms Parachlamydia acanthamoebae and Simkania negevensis. Results from this study confirm, on the one hand, that MOTT are probably the most important infectious etiology for granulomatous inflammation in reptiles. On the other hand, they indicate that chlamydia infects reptiles and that Cp. pneumoniae should be considered an etiological agent of granulomatous lesions of reptiles. Because both MOTT and Cp. pneumoniae are human pathogens, the potential of zoonotic transmission from reptiles to humans has to be considered. In contrast, the significance of Chlamydia-like isolates remains completely open, and further studies are needed to evaluate their role.