Characterization of multidrug-resistant avian pathogenic Escherichia coli: an outbreak in canaries.

The canary (Serinus canaria) is appreciated for its beautiful song, colors, and docile temperament and drives a lucrative business. However, diseases caused by avian pathogenic Escherichia coli (APEC) compromise the health of canaries, and the inadequate antimicrobial treatment can lead to the emergence of resistant strains. This study aimed to characterize 21 isolates of E. coli obtained from canaries infected with colibacillosis during an outbreak in northern Paraná State, Brazil. APEC and diarrheagenic E. coli (DEC) virulence genes were screened for by polymerase chain reaction (PCR). All isolates were positive for the hlyF, iss, and ompT genes, which are characteristic of APEC. The iroN gene was found in 95.2% of isolates, and none had the iutA gene. The ipaH gene, characteristic of enteroinvasive E. coli (EIEC), was found in 71.4% of isolates, all belonging to the phylogenetic group B1. High genetic similarity (>95%) was found using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The isolates belonged to serotypes O117:H4 (71.4%) and O1:H20 (23.8%). This is the first report of a clonal colibacillosis outbreak in canaries caused by APEC. All isolates were resistant to ampicillin, nalidixic acid, ciprofloxacin, enrofloxacin, norfloxacin, and tetracycline. The high rate of multidrug resistance in our study shows the importance of avoiding the inadequate antibiotic treatment. We suggest that further studies should be conducted to contribute to the understanding of colibacillosis in canaries since the health of animals is linked to human and environmental health, as defined by the concept of One Health.

Detection of Enterobacteriaceae, antimicrobial susceptibility, and virulence genes of Escherichia coli in canaries (Serinus canaria) in northeastern Brazil / Detecção de enterobactérias, sensibilidade antimicrobiana e genes de virulência de Escherichia coli em canários belgas (Serinus canaria) da região Nordeste do Brasil

This study aimed to verify the presence of members from the Enterobacteriaceae family and determine antimicrobial susceptibility profiles of the isolates in canaries bred in northeastern Brazil; in addition, the presence of diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC) was also verified in these birds. Samples were collected during an exhibition organized by the Brazilian Ornithological Federation in July 2015 in Fortaleza, Brazil. A total of 88 fecal samples were collected and submitted to pre-enrichment step using buffered peptone water, followed by enrichment with the following broths: brain-heart infusion, Rappaport-Vassiliadis, and Selenite-Cystine. Subsequently, aliquots were streaked on MacConkey, brilliant green and salmonella-shigella agar plates. Colonies were selected according to morphological characteristics and submitted to biochemical identification and antimicrobial susceptibility tests with disk-diffusion technique. E. coli strains were evaluated for the presence of eight DEC genes and five APEC genes through conventional polymerase chain reaction (PCR) screening. The most frequent species observed were Pantoea agglomerans (25%), Serratia liquefaciens (12.5%), and Enterobacter aerogenes (9.1%). A single rough strain of Salmonella enterica subsp. enterica was identified in one sample (1.1%). High resistance rates to amoxicillin (78.7%) and ampicillin (75.4%) were identified. Polymyxin B (9.8%), gentamycin (6.6%), and enrofloxacin (6.6%) were the most efficient antibiotics. The total number of multidrug-resistant strains (isolates resistant to more than three antimicrobial classes) was 23 (37.7%). Four E. coli strains were tested for the virulence genes, and two were positive for APEC virulence genes: one strain was positive for iutA and the other for hlyF. In conclusion, canaries in northeastern Brazil participating in exhibitions may present Salmonella spp., Escherichia coli and other enterobacteria in the intestinal microbiota with antimicrobial resistance. These results indicate that, although the E. coli strains recovered from canaries in this study have some virulence genes, they still do not fulfill all the requirements to be considered APEC.(AU)

O objetivo deste trabalho foi verificar a presença de enterobactérias e determinar o perfil de sensibilidade aos antimicrobianos dos isolados oriundos de canários belgas criados em cativeiro do Nordeste do Brasil, adicionalmente verificou-se a presença de Escherichia coli diarreiogênicas (DEC) e E. coli patogênica aviária (APEC) nesses animais. A colheita das amostras ocorreu durante uma exposição de canários belgas organizada pela Federação Ornitológica do Brasil (FOB), em julho de 2015, na cidade de Fortaleza, Ceará, Brasil. Um total de 88 amostras de fezes foram coletadas e submetidas a pré-enriquecimento utilizando água peptonada, caldo de enriquecimento Brain Heart Infusion, Rappaport-Vassiliadis e Selenito-Cistina. Fez-se triagem em placas de ágar MacConkey, Verde Brilhante e ágar Salmonella Shigella. As colônias foram selecionadas e submetidas à identificação bioquímica e susceptibilidade antimicrobiana. Estirpes de Escherichia coli foram avaliadas quanto a presença de 8 genes de virulência de DEC e cinco de APEC por reação em cadeia da polimerase convencional (PCR). As enterobactérias encontradas com maior frequência foram Pantoea agglomerans (25%), Serratia liquefaciens (12,5%) e Enterobacter aerogenes (9,1%). Uma única estirpe de Salmonella enterica subsp. enterica (rugosa) esteve presente em um dos isolados (1,1%). Altos percentuais de resistência foram encontrados para dois antibióticos: amoxicilina (78,7%) e ampicilina (75,4%). Polimixina B (9,8%), gentamicina (6,8%) e enrofloxacina (6,5%) foram os antibióticos com melhor eficiência. O total de estirpes multirresistentes (a mais de três classes de antimicrobianos) foi de 23 (37,7%). Das quatro estirpes de E. coli isoladas, duas foram positivas para os genes de APEC, sendo uma estipe para o gene iss e outra para os genes iutA e hlyF. Portanto, canários belgas criados em cativeiro no Brasil que participam de exposições podem apresentar Salmonella spp., Escherichia coli e outras enterobactérias em sua microbiota intestinal com resistência antimicrobiana. Estes resultados indicam que as estirpes de E. coli isoladas de canário belga no presente estudo apresentam alguns, mas não todos, genes de virulência para serem caracterizadas como E. coli patogênica para aves (APEC).(AU)

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR < 0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR < 0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community. HIGHLIGHTS Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds. Differences were found between infected and healthy animals in gut microbiota. Candidatus Arthromitus was closely associated with infected birds. M. ornithogaster can affect intestinal microbiota composition of canaries.

Reproductive disorders in domestic canaries (Serinus canarius domesticus): a retrospective study on bacterial isolates and their antimicrobial resistance in Italy from 2009 to 2012.

Reproductive disorders are responsible for significant economic losses in canary aviculture due to embryo and newborn chick mortality. Most of the time, deaths are caused by bacterial pathogens, however little published data exist about the prevalence of bacterial isolates that are identified during diagnostic protocols. This study reports on data collected from previous investigations carried out on cloacal swabs (n 456), unhatched eggs (n 52), and dead newborn chicks (n 68) collected from canary aviaries with a history of reproductive disorders. Of the examined samples, 41% were positive for the presence of pathogenic or potentially pathogenic bacteria, with particular regard to Gram negative species during bacteriological investigations. The most prevalent microorganisms were Gram-negative (55%). A predominance of Klebsiella, Escherichia, and Pantoea genera was observed. These are usually associated to pathological conditions in pet birds. Among Gram-positive bacteria, Bacillus spp. and Staphylococcus spp. were most prevalent. The antimicrobial susceptibility testing carried out on bacterial isolates showed a multiple resistance, especially against amoxycillin, erythromycin, spiramycin, tiamulin, and tylosin. This study represents a first attempt to provide an update on microbial causes of embryonic and neonatal mortality in canary aviaries in Italy; in addition, it provides further understandings about the efficacy of antimicrobial therapy.

A survey on gram-negative bacteria in saffron finches (Sicalis flaveola) from illegal wildlife tradein Brazil / Pesquisa de bactérias gram-negativas em canários-da-terra (Sicalis flaveola) resgatados do tráfico ilegal de aves no Brasil

Passerines such as canaries or finches are the most unlawfully captured species that are sent to wildlife centers in São Paulo, Brazil. Captured birds may have infection by opportunistic bacteria in stressful situations. This fact becomes relevant when seized passerine are reintroduced. The aim of this study was to evaluate the health state of finches from illegal wildlife trade using microbiological approaches. Microbiological samples were collected by cloacal and tracheal swabs of 100 birds, captured during 2012 and 2013. The results indicate high frequency of gram-negative bacteria in feces and oropharynx, especially from the Enterobacteriaceae family (97.5%). The most frequent genera were Escherichia coli (46.5%) and Klebsiella pneumoniae (10.4%). Enterobacter cloacae, Serratia liquefaciens, Serratia spp. Klebsiella oxytoca and Citrobacter freundii were isolated with lower frequency from asymptomatic birds. The presence of enteropathogenic Escherichia coli (EPEC) and Shiga toxinproducing strain (STEC) confirm the zoonotic risks and public health concern.(AU)

No Estado de São Paulo, Brasil, os pássaros como os canários-da-terra têm sido uma das espécies mais frequentemente resgatadas do tráfico ilegal e enviadas aos centros de vida selvagem. Em situações de estresse estas aves podem ser acometidas por infecções causadas por bactérias oportunistas. Este fato é de grande importância quando é planejada da reintrodução das aves na natureza. O presente trabalho foi delineado para avaliar o estado de saúde de canários-da-terra resgatados do tráfico ilegal. Foram colhidas soabes da traqueia e da cloaca de 100 aves resgatadas durante os anos de 2012 e 2013. Os resultados obtidos revelaram alta frequência de bactérias gram-negativas nas fezes e no orofaringe dos animais, com maior frequência para os membros da família Enterobacteriaceae (97,5%). Os gêneros mais frequentes foram Escherichia coli (46,55) e Klebsiella pneumoniae (10,4%). Outros microorganismos incluindo Enterobacter cloacae, Serratia liquefaciens, Serratia spp, Klebsiella oxytoca e Citrobacter freundii também foram isolados em menor frequencia de aves assintomáticas. A presença de estirpes de Escherichia coli enteropagênicas (EPEC) e as produtoras da toxina de Shiga confirmam o risco de zoonose e a importância para saúde pública deste tipo de ave.(AU)

Identification and antimicrobial resistance of members from the Enterobacteriaceae family isolated from canaries (Serinus canaria) / Identificação e resistência antimicrobiana de membros da família Enterobacteriaceae isolados de canários (Serinus canaria)

The Enterobacteriaceae family contains potentially zoonotic bacteria, and their presence in canaries is often reported, though the current status of these in bird flocks is unknown. Therefore, this study aimed to identify the most common genera of enterobacteria from canaries (Serinus canaria) and their antimicrobial resistance profiles. From February to June of 2013, a total of 387 cloacal swab samples from eight domiciliary breeding locations of Fortaleza city, Brazil, were collected and 58 necropsies were performed in canaries, which belonged to the Laboratory of Ornithological Studies. The samples were submitted to microbiological procedure using buffered peptone water and MacConkey agar. Colonies were selected according to their morphological characteristics on selective agar and submitted for biochemical identification and antimicrobial susceptibility. A total of 61 isolates were obtained, of which 42 were from cloacal swabs and 19 from necropsies. The most isolated bacteria was Escherichia coli with twenty five strains, followed by fourteen Klebsiellaspp., twelve Enterobacterspp., seven Pantoea agglomerans, two Serratiaspp. and one Proteus mirabilis. The antimicrobial to which the strains presented most resistance was sulfonamides with 55.7%, followed by ampicillin with 54.1% and tetracycline with 39.3%. The total of multidrug-resistant bacteria (MDR) was 34 (55.7%). In conclusion, canaries harbor members of the Enterobacteriaceae family and common strains present a high antimicrobial resistance rate, with a high frequency of MDR bacteria.

A família Enterobacteriaceae possui bactérias com potencial zoonótico e a presença destas bactérias em canários é relatada na literatura, porém a realidade dos plantéis de criadores de canários é desconhecida. Portanto, este trabalho teve como objetivo isolar enterobactérias de canários belga (Serinus canarius) com o intuito de conhecer os gêneros mais comuns nestas aves e suas respectivas resistências a antimicrobianos. De fevereiro a junho de 2013 foram coletadas 387 amostras de swabs cloacais de canários de oito propriedades da cidade de Fortaleza, Brasil e de 58 necropsias de aves do acervo próprio do Laboratório de Estudos Ornitológicos. As amostras foram submetidas a isolamento microbiológico utilizando-se água peptonada e ágar MacConkey. As colônias foram selecionadas de acordo com suas características morfológicas nas placas, submetidas à tipificação bioquímica para identificação e ao teste de sensibilidade a antimicrobianos. Foram isoladas 61 cepas, sendo 42 de suabes cloacais e 19 de necropsias. A bactéria mais isolada foi Escherichia coli com vinte e cinco cepas, seguida por catorze Klebsiella spp., doze Enterobacter spp., sete Pantoea agglomerans, duas Serratiaspp. e uma cepa de Proteus mirabilis. As cepas apresentaram maior resistência a sulfonamidas com 55,7%, seguidas por ampicilina com 54,1% e tetraciclina com 39,3%. Além disso, o total de cepas resistentes a múltiplas drogas (RMD) foi 34 (55,7%). Portanto, conclui-se que os canários albergam enterobactérias e que as cepas apresentam alto índice de resistência a antimicrobianos, com alta frequência de cepas RMD.

Identification of shiga toxin and intimin genes in Escherichia coli detected from canary (Serinus canaria domestica).

The pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains is, in large part, due to shiga toxin (Stx) genes (Stx1 and Stx2) and/or intimin (eae) gene. The purpose of this study was to analyze the role of domestic canaries (Serinus canaria domestica) as a reservoir of Stx and intimin producing strains of E. coli. For this study, a total of 50 cloacal swabs were collected from 50 healthy domestic canaries. Cloacal swabs were cultured and tested using standard methods of microbiology. After primary identification of E. coli, DNA was extracted and polymerase chain reaction was performed using specific primers for Stx1, Stx2 and eae genes. In this study, three of 50 samples were found to be Stx2 positive. In the present study, nine (18%) of 50 canaries tested were positive for eae gene. Only 2% of total canaries tested were positive for simultaneous Stx and eae genes. By considering the presence of Stx genes in E. coli isolated from cloacal contents of canary, this hypothesis expressed that the canaries may be the carriers of virulence genes that can risk human health. Canary was considered to be a reservoir of Stx and intimin genes and make these birds important vehicles for the spread of zoonosis infection.

Prolonged fecal shedding of 'megabacteria' (Macrorhabdus ornithogaster) by clinically healthy canaries (Serinus canaria).

Macrorhabdus ornithogaster, often referred to as 'megabacterium', is an ascomycetous yeast usually found colonizing the mucosal surface of the isthmus existing between the glandular and grinding stomach of a wide diversity of bird species. However, this yeast can also behave as an avian pathogen, therefore representing a potential threat to bird breeding. The aim of this work was to assess the prevalence and patterns of fecal shedding of M. ornithogaster in a colony of healthy canary birds (Serinus canaria) bred in captivity. Fresh fecal samples from 39 canaries (17 males and 22 females) were cultured in liquid media for M. ornithogaster enrichment. Only two clinically healthy females were fecal culture-positive for the yeast, which represents an overall prevalence of 5.13% in the sampled population. A close surveillance of the two culture-positive canaries, which included periodical microscopic examination of fresh stool samples, showed prolonged fecal shedding of M. ornithogaster. Nevertheless, both animals remained asymptomatic throughout the study period. To the best of our knowledge, this is the first study reporting the continuous shedding of M. ornithogaster by clinically healthy canaries.

Chlamydia psittaci infection in canaries heavily infested by Dermanyssus gallinae.

Dermanyssus gallinae is a haematophagous ectoparasite responsible for anemia, weight loss, dermatitis and a decrease in egg production. Dermanyssus gallinae may play a role in the modulation of the host immune system, maybe predisposing the host to some bacterial infections such as chlamydiosis. This is an important zoonosis. Humans are exposed to Chlamydia psittaci through inhalation of the agent dispersed from the infected birds. In this study, a syndrome observed in an aviary of canaries was investigated. A heavy infestation by D. gallinae was reported. Simultaneously, a C. psittaci infection was molecularly confirmed in the canaries. Combined therapy was applied successfully. The association of C. psittaci with the examined mites has been confirmed. Therefore, we think that D. gallinae have played a role in the spreading of C. psittaci infection among the canaries. Moreover, D. gallinae could have played an important role predisposing the canaries to the development of chlamydiosis, by inducing anemia and debilitation. The control of mites in the aviaries may represent a crucial step for the prevention of important infection such as chlamydiosis in birds and humans.

Systemic listeriosis in caged canaries (Serinus canarius).

The occurrence of listeriosis in 12 caged canaries is described where 50% of the birds, including the female and all of the offspring, died within 2 weeks without clinical signs. At necropsy, multifocal necrotizing and partly granulomatous hepatitis, splenitis, myocarditis, interstitial nephritis, and exudative pericarditis with intra-lesional Listeria monocytogenes were the predominant findings as shown by histopathology and immunohistochemistry. Microbiology, serology and polymerase chain reaction revealed L. monocytogenes serotype 1/2a as the causative agent. Thus listeriosis has to be considered in the differential diagnosis for granulomas associated with mycobacteriosis, yersiniosis, coligranulomatosis or fungal infections.

Streptococcus pluranimalium sp. nov., from cattle and other animals.

Strains from subclinical mastitis, from the genital tract and tonsils of cattle, from tonsils of a goat and a cat and from the crop and the respiratory tract of canaries were found to constitute a new streptococcal species, for which the name Streptococcus pluranimalium sp. nov. is proposed. Sequencing of 16S rRNA showed that Streptococcus thoraltensis and Streptococcus hyovaginalis were its closest known phylogenetic relatives. The new species showed some phenotypic resemblance to the poorly described species Streptococcus acidominimus, but whole-cell protein analysis and 16S rRNA sequencing revealed that the new species was only distantly related to the type strain of S. acidominimus. Identification of these bacteria, which showed heterogeneous biochemical reaction patterns, was most reliably made by whole-cell protein analysis. Nevertheless, a number of biochemical reactions can be used to differentiate S. pluranimalium from other animal streptococci. Strain LMG 14177T, isolated from mastitic milk of a dairy cow, was designated as the type strain of S. pluranimalium sp. nov.

Fulminant psittacosis requiring mechanical ventilation and demonstrating serological cross-reactivity between Legionella longbeachae and Chlamydia psittaci.

Chlamydia psittaci infection typically causes a mild respiratory illness in humans. Severe respiratory failure requiring mechanical ventilation or intensive care therapy is an uncommon development. The aetiological agents causing severe community acquired pneumonia often remain undetermined. Serological tests may aid in diagnosis. We present two cases of fulminant psittacosis, one demonstrating early cross-reactivity with Legionella longbeachae.

Killer activity at different pHs against Cryptococcus neoformans var. neoformans serotype A by environmental yeast isolates.

Yeast isolates that share the same habitats as Cryptococcus neoformans var. neoformans serotype A in a restricted Mediterranean area were assayed in order to verify their killer activity against Cr. neoformans strains isolated from clinical and environmental sources. Many of the environmental yeast isolates expressed the killer phenomenon against the assayed strains of Cr. neoformans. Two species of Candida: Candida parapsilosis and Candida famata, and Pichia carsonii, were the most active killers at pH 4.6, 5.0 and 5.6 levels encountered in pigeon and canary guanos. Killer activity by C. parapsilosis is reported for the first time. The authors hypothesized that the killer phenomenon exerted by yeast species with heavy killer activity against Cr. neoformans would lend themselves for use as biological control agents against sensitive strains of Cr. neoformans when directly inoculated into the habitats of Cr. neoformans.

Serotyping of Cryptococcus neoformans isolates from environmental and clinical sources in extreme southern Italy (Calabria and Sicily, central Mediterranean area).

Ninety-seven environmental and four clinical isolates of Cryptococcus neoformans from several localities in extreme southern Italy were serotyped. All proved to be Cr. neoformans var. neoformans serotype A. The homogeneity of the serotypes suggests that geographical climatological conditions may play a role in Cr. neoformans serotype diffusion. It was furthermore revealed that, unlike the data reported for the serotypes of Cr. neoformans in mainland Italy, Cr. neoformans serotype D was not present in the examined sites in extreme southern Italy.

Mycobacterium genavense infection in canaries.

A case of mycobacteriosis in a collection of canaries (Serinus canarius) is described. The affected birds showed nonspecific clinical signs and macroscopic lesions (slight splenomegaly). Histologic lesions found in liver, spleen, lungs, and kidneys consisted of noncaseous nodules containing accumulations of large macrophages that showed a highly vacuolated cytoplasm in which numerous acid-fast organisms were detected. Attempts to isolate and culture the organisms using Löwenstein-Jensen and Coletsos media proved unsuccessful. Microorganisms belonging to the species Mycobacterium genavense were identified by means of polymerase chain reaction techniques in hepatic tissue from both birds. This finding confirms the existence of fastidiously growing mycobacterial infections, other than Mycobacterium avium, in birds.

Evidence of canary droppings as an important reservoir of Cryptococcus neoformans.

After preliminary results had shown the occurrence of Cryptococcus neoformans in canary droppings in southern Italy, the increasing epidemiological interest in cryptococcosis and the habitats of the agent led to more extensive studies. 180 samples of canary droppings were collected from pet shops and private households in two towns (Messina and Reggio Calabria) of southern Italy and culturally examined for C. neoformans. The examination was carried out with the help of the brown colour effect (BCE) specific of the C. neoformans colony on Guizotia abyssinica creatinine agar (Staib agar). 48 samples (26.6%) were positive; of these, 32/108 (29.6%) were from pet shops and 16/72 (22.2%) from private households. The colony growth was not mucous, i.e. microscopically the blastospores were found to be little or not encapsulated. All C. neoformans strains tested by the auxanographic method showed a weak assimilation of creatinine. The variety status of the isolates was tested with canavanine-glycine-bromothymol-blue (CGB) agar; all strains tested (n = 420) were found to be C. neoformans var. neoformans. In the light of extensive studies on C. neoformans and the canary in the pre-AIDS era in Germany, the present observations in Italy ask for further basic research, epidemiological studies, and education of the public.

Host restriction in the productive cycle of avian polyomavirus budgerigar fledgling disease virus type 3 depends on a single amino acid change in the common region of structural proteins VP2/VP3.

The three avian polyomaviruses budgerigar fledgling disease virus types 1 to 3 (BFDV-1 to -3) contain genomes of identical size, 4981 bp. With differences of up to only 15 bp between the three genomes, these viruses show distinct tropism for cultured cells of various avian species: infection of chicken embryo (CE) cells with BFDV-1 and -2 results in virus propagation, whereas BFDV-3 is not replicated; all three viruses replicate, with different efficiencies, in infected Muscovy duck cells. Transfection of CE cells with BFDV-3 DNA results in a single productive cycle. As shown by construction of hybrid virus genomes and site-directed mutagenesis, a single amino acid difference (glycine instead of valine or alanine) within the common region of the minor structural proteins VP2/VP3 is responsible for this type of abortive infection of CE cells. Further experiments indicate a defect in one of the early steps during infection, at or prior to uncoating.