Human Conjunctivitis Research Articles

Zoonotic transmission of Chlamydia felis from domestic cats; A case series of chronic follicular conjunctivitis in humans

We present three cases of chronic follicular conjunctivitis resulting from the zoonotic transmission of Chlamydia felis from domestic cats. Our objective is to raise awareness regarding the potential zoonotic transmission of Chlamydia felis from domestic cats and describe the methodology for definitive pathogen identification using Polymerase Chain Reaction (PCR) and subsequent sequence analysis, a useful tool in the identification of these rare pathogens. We discuss the factors that could be contributing to the potential under-diagnosis of zoonotic C. felis infections and propose a treatment regime for cases of C. felis-related conjunctivitis.

Newcastle Disease - overview and analysis of the current spread of the disease in Bulgaria and Europe

Newcastle disease (ND) is a highly contagious and severe worldwide disease affecting birds. The infection is a mild zoonosis and can cause conjunctivitis in humans, but the condition is usually mild and self-limiting. ND is caused by virulent strains of avian paramyxovirus type 1. The disease occurs in five forms: velogenic viscerotrophic, velogenic neutrotropic, mesogenic, lentogenic Newcastle disease and asymptomatic enteric Newcastle disease. Lentogenic strains are very widespread, but are usually asymptomatic and do not cause outbreaks in poultry. Newcastle disease in its highly pathogenic form, is a WOAH listed disease and must be reported. It usually manifests as severe respiratory disease, depression, nervousness and/or diarrhea. The clinical picture is very similar to bird flu, so laboratory tests are important to confirm the diagnosis.

The structure and diversity of microbial communities in Paederus fuscipes (Coleoptera: Staphylinidae): from ecological paradigm to pathobiome

BackgroundPaederus fuscipes is medically the most famous rove beetle, which causes dermatitis or conjunctivitis in humans, as well as gastrointestinal toxicosis in livestock, via releasing toxic hemolymph containing pederin. Pedrin biosynthesis genes have been identified in uncultured Pseudomonas-like endosymbionts that are speculated to be acquired through a horizontal transfer. However, the composition of the P. fuscipes microbial community, especially of the gut and genital microbiome, remains unclear. This study was aimed to characterize the structure and diversity of P. fuscipes-associated bacterial communities in terms of gender, organ, and location using the Illumina HiSeq platform in the southern littorals of Caspian Sea.ResultsThe OTUs identified from P. fuscipes specimens were collapsed into 40 phyla, 112 classes, 249 orders, 365 families, 576 genera, and 106 species. The most abundant families were Pseudomonadaceae, Spiroplasmataceae, Weeksellaceae, Enterococcaceae, and Rhizobiaceae, respectively. Thirty top genera made up > 94% of the P. fuscipes microbiome, with predominating Pseudomonas, followed by the Spiroplasma, Apibacter, Enterococcus, Dysgonomonas, Sebaldella, Ruminococcus, and Wolbachia. Interesting dissimilarities were also discovered within and between the beetle microbiomes in terms of genders and organs. Analyses showed that Spiroplasma / Apibacter as well as Pseudomonas / Pseudomonas were the most abundant in the genitals / intestines of male and female beetles, respectively. Bacterial richness did not display any significant difference in the three provinces but was higher in male beetles than in females and more in the genitals than intestines.ConclusionsThe present study identified Pseudomonas-like endobacterium as a common symbiont of P. fuscipes beetles; this bacterium begins its journey from gut and genitalia of females to reach the male rove beetles. Additionally, male and female rove beetles were characterized by distinctive microbiota in different organs, likely reflecting different functions and/or adaptation processes. Evidence of the extension of P. fuscipes microbiome from the environmental paradigm to the pathobiome was also presented herein. A comprehensive survey of P. fuscipes microbiome components may eventually lead to ecological insights into the production and utilization of defensive compound of pederin and also the management of linear dermatitis with the use of available antibiotics against bacterial pathogens released by the beetles.AmgmLhee2SUeiaw49GR9ULVideo

Phylogeny and Density Dynamics of Wolbachia Infection of the Health Pest Paederus fuscipes Curtis (Coleoptera: Staphylinidae).

Simple SummaryWolbachia pipientis is a maternally inherited endosymbiont of arthropods and filarial nematodes, and was reported to occur in Paederus fuscipes, a beetle that causes dermatitis linearis and conjunctivitis in humans when they come in contact with skin. In this study, we report the phylogenetic position and density dynamics of Wolbachia in P. fuscipes. The phylogeny of Wolbachia, based on an analysis of MLST genotyping, showed that Wolbachia from P. fuscipes belongs to supergroup B. Quantitative PCR indicated that the infection density in adults was higher than in any other life stage (egg, larva or pupa), and that reproductive tissue in adults had the highest infection densities, with similar densities in the sexes. These findings provide a starting point for understanding Wolbachia infection dynamics in P. fuscipes, and interactions with other components of the microbiota.The maternally inherited obligate intracellular bacteria Wolbachia infects the reproductive tissues of a wide range of arthropods and affects host reproduction. Wolbachia is a credible biocontrol agent for reducing the impact of diseases associated with arthropod vectors. Paederus fuscipes is a small staphylinid beetle that causes dermatitis linearis and conjunctivitis in humans when they come into contact with skin. Wolbachia occur in this beetle, but their relatedness to other Wolbachia, their infection dynamics, and their potential host effects remain unknown. In this study, we report the phylogenetic position and density dynamics of Wolbachia in P. fuscipes. The phylogeny of Wolbachia based on an analysis of MLST genotyping showed that the bacteria from P. fuscipes belong to supergroup B. Quantitative PCR indicated that the infection density in adults was higher than in any other life stage (egg, larva or pupa), and that reproductive tissue in adults had the highest infection densities, with similar densities in the sexes. These findings provide a starting point for understanding the Wolbachia infection dynamics in P. fuscipes, and interactions with other components of the microbiota.

Pathogenesis, Transmissibility, and Tropism of a Highly Pathogenic Avian Influenza A(H7N7) Virus Associated With Human Conjunctivitis in Italy, 2013.

H7 subtype influenza viruses represent a persistent public health threat because of their continued detection in poultry and ability to cause human infection. An outbreak of highly pathogenic avian influenza H7N7 virus in Italy during 2013 resulted in 3 cases of human conjunctivitis. We determined the pathogenicity and transmissibility of influenza A/Italy/3/2013 virus in mouse and ferret models and examined the replication kinetics of this virus in several human epithelial cell types. The moderate virulence observed in mammalian models and the capacity for transmission in a direct contact model underscore the need for continued study of H7 subtype viruses.

Review on Newcastle Disease of Poultry and its Public Health Importance

Newcastle disease is a contagious bird disease affecting many domestic and wild avian species and which can be transmissible to humans. It is caused by avian paramyxovirus serotype 1 virus which, with viruses of the other eight serotypes (avian paramyxovirus 1-9) has been placed in the genus Avulavirus, sub-family Paramyxovirinae, family Paramyxoviridae. Virulent Newcastle disease virus strains are endemic in poultry in most of Asia, Africa, and some countries of North and South America. Other countries, including the united states of America and Canada, are free of those strains in poultry. Highest prevalence of Newcastle disease is recorded in cross breeds of chickens than local breed and the low altitudes do have higher prevalence than the mid and high altitudes. The transmission of Newcastle disease occurs through respiratory aerosols, exposure to fecal and other excretions from infected birds, through newly introduced birds, selling and giving away sick birds and contacts with contaminated feed, water, equipment and clothing. The strain of Newcastle pathogenicity can be classified into five pathotype: Asymptomatic enteric strain; Lentogenic strain; Mesogenic stain; Viscerotropic velogenic strain and Neurotropic velogenic strain. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and preexisting immunity caused by paramixovirus with worldwide distribution affecting chickens (all poultry and birds are susceptible) of All age group are susceptible. Symptoms from the respiratory tract are gasping, coughing, sneezing and rales. Signs from the nervous system include tremors, paralyzed wings and legs, twisted necks, circling, clonic spasms and sometimes complete paralysis. Other general symptoms that can be seen are greenish diarrhea, depression and inappetence, partial or complete drop in egg production and an increased production of deformed eggs. Clinical diagnosis based on history, signs and lesions may establish a strong index of suspicion but the laboratory confirmation must be done. The general approaches to the control of Newcastle disease are hygiene and vaccination. Humans are among the many species that can be infected by Newcastle disease in addition to avian species. Newcastle disease may cause conjunctivitis in humans, when a person has been exposed to large quantities of the virus. Mostly, Laboratory workers and vaccinators are affected. Recently, the disease which decreases the development of poultry production for industry is the infectious diseases, among infection disease Newcastle is the one which causes economical lose of poultry and its product. The objective of this review is to understand the Newcastle disease causative agent, pathogenicity, clinical sign and how to prevent and control the Newcastle disease, which concerned with the currently published or reported research.

Carbohydrate Determinants in Ferret Conjunctiva are Affected by Infection with Influenza H1N1 Virus

Background: Carbohydrates often accomplish as cell-surface receptors for microorganisms and influenza virus preferentially binds to sialic acid through the viral haemagglutinin. The virus may attach not only to the epithelium in the airways, but also to the surface ocular epithelium.Purpose: To decide if ferrets can be used to study virus induced conjunctivitis and to evaluate changes in the conjunctival glycosylation pattern during an influenza attack.Methods: Ferrets were infected with H1N1 influenza virus via nasal inoculation. The in situ carbohydrate expressions in eyelid sections from ferrets 0 to 10 days after infection was examined using lectin- and immunohistochemistry.Results: The conjunctival cells became hypertrophic with appearance of both PAS positive and PAS + Alcian Blue stained cells 5–6 days after inoculation. The binding of three sialic acid detecting lectins were investigated: WGA, MAA2 and SNA1. While none of them stained conjunctival epithelial cells in the non-infected ferrets to any extent, there was a positive conjunctival reaction in the infected ferret after incubation with all three lectins. Binding of a MUC1 antibody that seems to detect sialylated determinants in the mucin molecule indicates that MUC1 is de novo expressed in most of the squamous conjunctival cells at the start of the influenza infection. MUC5AC positive epithelial cells, probably goblet cells, proliferate in the diseased conjunctiva.Conclusion: Nasal inoculation of H1N1 virus to ferrets has an effect on the conjunctival cells and change their expression of glycans. Synthesized glycans are an integral part of the tear film and the present study contributes to reveal the changes that occur in the surface epithelium in the eyelid and thereby to elucidate the pathophysiology of the virus mediated conjunctivitis. Ferrets are suitable animal models to study human conjunctivitis mediated by human influenza virus.

Pathogenesis, Transmissibility, and Ocular Tropism of a Highly Pathogenic Avian Influenza A (H7N3) Virus Associated with Human Conjunctivitis

H7 subtype influenza A viruses, responsible for numerous outbreaks in land-based poultry in Europe and the Americas, have caused over 100 cases of confirmed or presumed human infection over the last decade. The emergence of a highly pathogenic avian influenza H7N3 virus in poultry throughout the state of Jalisco, Mexico, resulting in two cases of human infection, prompted us to examine the virulence of this virus (A/Mexico/InDRE7218/2012 [MX/7218]) and related avian H7 subtype viruses in mouse and ferret models. Several high- and low-pathogenicity H7N3 and H7N9 viruses replicated efficiently in the respiratory tract of mice without prior adaptation following intranasal inoculation, but only MX/7218 virus caused lethal disease in this species. H7N3 and H7N9 viruses were also detected in the mouse eye following ocular inoculation. Virus from both H7N3 and H7N9 subtypes replicated efficiently in the upper and lower respiratory tracts of ferrets; however, only MX/7218 virus infection caused clinical signs and symptoms and was capable of transmission to naive ferrets in a direct-contact model. Similar to other highly pathogenic H7 viruses, MX/7218 replicated to high titers in human bronchial epithelial cells, yet it downregulated numerous genes related to NF-κB-mediated signaling transduction. These findings indicate that the recently isolated North American lineage H7 subtype virus associated with human conjunctivitis is capable of causing severe disease in mice and spreading to naive-contact ferrets, while concurrently retaining the ability to replicate within ocular tissue and allowing the eye to serve as a portal of entry.

The efficacy of stingless bee honey for the treatment of bacteria-induced conjunctivitis in guinea pigs.

The efficacy of the conjunctival application of a crude concentration of stingless bee honey (SBH) for the treatment of bacterial conjunctivitis was investigated in an animal model. Bacterial conjunctivitis caused by Staphylococcus aureus or Pseudomonas aeruginosa was induced in Hartley guinea pigs. The conjunctival application of SBH or gentamicin was used for treatment, and the results of this treatment were compared with control values. Inflammatory signs, duration of infection (ie, positive culture), and time for the complete resolution of infection with S. aureus or P. aeruginosa were shortened by the conjunctival application of 1 drop (70 µL) of crude SBH twice daily. The potency of SBH was comparable with that of gentamicin. SBH may be a rational agent for the treatment of infective conjunctivitis in humans; it is inexpensive and commonly available to the rural population.

Traditional Japanese Herbal (Kampo) Medicines and Treatment of Ocular Diseases: A Review

Herbal medicines have been used clinically in Eastern Asia, and traditional Japanese herbal (Kampo) formulas are approved as ethical drugs. The Kampo formulas are mixtures of the crude extracts of several herbs, each of which contains multiple components. Numerous investigators have reported that some herbal medicines are efficacious for treating several human diseases. We reviewed the literature on traditional herbal medicines and treatment of ocular diseases. Oral Orengedoku-to and Kakkon-to inhibit postoperative uveitis in humans. Oral Goshajinki-gan improved ocular surface disorders in patients with type 1 diabetes mellitus. Oral Hachimijio-gan increased retinal blood flow. Keishi-bukuryo-gan Sho might be associated with vitreoretinopathy in patients with type 2 diabetes mellitus. Oral Hachimijio-gan and Goshajinki-gan delayed lens opacification in rats and mice. Oral Sairei-to, Orengedoku-to, Senkanmeimoku-to, Scutellariae radix extract, Gardeniae fructus extract, topical Liguisticum wallichii rhizoma extract, and intravenous injection of tetramethylpyrazine, baicalin, baicalein, wogonin, and crocetin inhibited some forms of experimental uveitis in rabbits. Topical glycyrrhizinate improved allergic conjunctivitis in humans and rats. Oral crocetin improved eyestrain in humans. Oral berberine diminished experimental uveitis in rats. Baicalein, wogonin, berberine, and berberrubine inhibited in vitro expression of several cytokines in cultured retinal pigment epithelial cells. Some Kampo formulas are efficacious for treating several ocular diseases in humans and animals. Some herbal extracts and their components inhibit some forms of experimental uveitis.

Moraxella lacunata Infective Endocarditis after Tattooing as Confirmed by 16S rRNA Gene Sequencing from Heart Valve Tissue

0196-4399/00 (see frontmatter) © 2010 Elsevier Clinical Microbiology Newsletter 32:1,2010 Introduction Members of the genusMoraxella are normal commensals of the human upper respiratory tract and are occasionally recovered from the skin and mucous membranes.Moraxella lacunata has been associated with human conjunctivitis and keratitis but rarely with invasive and serious infections (1). However, a few case reports have established that M. lacunata organisms cause invasive infections in humans, including infective endocarditis (IE) (1-3), septicemia, septic arthritis, spondylodiscitis, and meningitis (4). In this report, we describe a patient who, after receiving a tattoo, developedM. lacunata endocarditis with cardiovascular complications, resulting in valve replacement surgery. This is the first report in Latin America of M. lacunata-induced endocarditis confirmed by sequencing of the 16s rRNA gene directly from heart valve tissue.

Chlamydophila felisCF0218 Is a Novel TMH Family Protein with Potential as a Diagnostic Antigen for Diagnosis ofC. felisInfection

Chlamydophila felis is a causative agent of acute and chronic conjunctivitis and pneumonia in cats (feline chlamydiosis). Also, C. felis is a suspected zoonotic agent of such diseases as non-Chlamydia trachomatis conjunctivitis in humans, although this is controversial. At present, there is no serodiagnostic system that specifically detects C. felis infection conveniently. Current systems use antigens such as lipopolysaccharide that cross-react with all chlamydia species. In addition, it is difficult to distinguish between cats that are vaccinated with the commercial vaccine against C. felis and cats that are infected with C. felis. Here, we describe a new candidate diagnostic antigen for diagnosis of C. felis infection, CF0218, that was obtained by screening a genomic expression library of C. felis Fe/C-56 with C. felis-immunized serum. CF0218 was a putative transmembrane head (TMH) family protein with bilobed hydrophobic motifs at its N terminus, and orthologues of CF0218 were not found in the Chlamydophila pneumoniae or Chlamydia trachomatis genomes. The recombinant CF0218 was not recognized by antiserum against C. trachomatis, suggesting that CF0218 is C. felis specific. CF0218 transcription during the course of C. felis infection was confirmed by reverse transcription-PCR. By indirect immunofluorescence analysis, CF0218 was colocalized with the C. felis-formed inclusion bodies in the infected cells. The antibody response against CF0218 was elevated following C. felis infection but not by vaccination in experimentally vaccinated and infected cats. These results suggest that CF0218, a novel TMH family protein of C. felis, possesses potential as a C. felis infection-specific diagnostic antigen.

Avian influenza A/(H7N2) outbreak in the United Kingdom

Several cases of influenza-like-illness (ILI) and/or conjunctivitis in humans have been linked to an outbreak of avian influenza in poultry at a smallholding near Corwen in northern Wales in the United Kingdom (UK).

Genotypes and Haplotypes of CCR2 and CCR3 Genes in Japanese Cedar Pollinosis

Whole genome scan analyses have revealed that the chromosomal region 3p21.3, which contains a gene cluster of the CC chemokine receptor, is possibly critical for the pathogenesis of allergic inflammation. Japanese cedar pollinosis is mediated by a type I allergy and induces seasonal rhinitis and conjunctivitis in humans as the most common form of hay fever in spring in Japan, although the candidate genes for cedar pollinosis remain to be elucidated. We sequenced CCR1, CCR2, CCR3, CCR5, and CCXCR1 using the PCR restriction fragment length polymorphism method in subjects with cedar pollinosis and controls. We found 8 polymorphisms of A111G, Arg127Cys and Arg252Gln in CCXCR1, T885C in CCR1, Val64Ile and T780C in CCR2, T51C in CCR3 and Arg223Gln in CCR5. The transmission disequilibrium test using 60 children with pollinosis and their parents and an association study using unrelated adult subjects (151 patients and 157 controls) showed a significant association of 64Ile in CCR2 and 51C in CCR3 with cedar pollinosis. The frequency of haplotype 64Ile/780C/51C in pollinosis was significantly higher than in controls. Our results suggest that CCR2 and CCR3 genes are candidate genes for Japanese cedar pollinosis.

Outbreak of low pathogenicity H7N3 avian influenza in UK, including associated case of human conjunctivitis

On 26 April 2006, the veterinary authorities in the United Kingdom reported that there had been an outbreak of avian influenza type A/H7 among a 35 000-bird housed poultry flock in eastern England, United Kingdom.

Cytokine profiles of peripheral blood mononuclear cells from dogs experimentally sensitized to Japanese cedar pollen

Japanese cedar ( Cryptomeria japonica, CJ) pollinosis is mediated by type-I hypersensitivity and induces seasonal rhinitis and conjunctivitis in humans. Previous studies showed that dogs could be experimentally sensitized with CJ pollen. In this study, we carried out quantitative analysis of mRNA levels of various cytokines in the peripheral blood mononuclear cells (PBMC) of 12 dogs experimentally sensitized to Japanese cedar pollen. Experimental sensitization was carried out by injection of crude CJ pollen extract with aluminium hydroxide gel. The expression levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, interferon (IFN)-γ, transforming growth factor (TGF)-β 1, and tumor necrosis factor (TNF)-α mRNAs in the PBMC were quantified using a real-time sequence detection system. In the PBMC tested without culture, the expression levels of IL-8 and TNF-α mRNAs in experimentally sensitized dogs were significantly higher than those in control dogs. The expression level of IFN-γ mRNA in the sensitized group was significantly lower than that in the control group. When the PBMCs were cultured in the presence of CJ pollen extract, the level of IL-4 mRNA expression was markedly increased in the PBMC from the experimentally sensitized dogs. In the PBMC stimulated with the CJ pollen extract, the expression level of IL-2 mRNA in the sensitized group was also significantly higher than that in the control group. Our data indicated that a Th2 response and proliferation of PBMC occur in response to the sensitizing antigen in dogs experimentally sensitized with CJ pollen, and revealed the presence of antigen-specific Th2 cells in this canine model. In addition, the expression levels of the mRNAs encoding proinflammatory cytokines were shown to be elevated after CJ pollen sensitization, indicating the activation of monocytes and macrophages.

Characterization of Cytokine mRNA Transcripts in Conjunctival Cells from Patients with Allergic Conjunctivitis

The host response to allergens appears to be regulated by specific patterns of local cytokine production. More than 20,000 conjunctival superficial cells were collected with a special brush, a smaller version of the Cytobrush used in cervical cytology, from the upper palpebral conjuntiva.Samples were obtained by cytology brush from seven patients with allergic conjunctivitis and from seven healthy volunteers. Giemsa staining, immunocytochemistry, and flow cytometric analysis were performed. Cytokine gene expression was assayed by the reverse-transcription-polymerase chain reaction method.Giemsa staining of cytocentrifuged preparations from patients with allergic conjunctivitis showed conjunctival epithelial cells with lymphocytes, mast cells, and eosinophils. In an immunohistochemical study, a few CD3- and CD4-bearing cells, but not CD20- and CD14-bearing cells, were seen in patients. In 82.6 +/- 17% of the samples obtained from allergic patients, HLA-DR was present, but it was present in only 34.2 +/- 17.8% of samples from control subjects (P = 0.0001) using flow cytometric analysis. Steady state transcripts of mRNA for cytokines were analyzed with RT-PCR in conjunctival cell samples, and results showed that samples from allergic conjunctivitis expressed increased transcripts of interleukin 4 and interleukin 13 but virtually no interleukin 2 or interferon-gamma; six samples from seven healthy subjects expressed no interleukin 2, interleukin 4, interleukin 13, or interferon-gamma transcripts.These results suggest that the clinical features of allergic conjunctivitis in humans are associated with a specific local pattern of proinflammatory cytokine expression.

Protein antigens of Chlamydia psittaci present in infected cells but not detected in the infectious elementary body

Ocular infection of guinea pigs with the guinea pig inclusion conjunctivitis (GPIC) strain of Chlamydia psittaci produces a clinical condition representative of acute chlamydial conjunctivitis in humans. Guinea pigs which had recovered from two challenges with GPIC were used as a source of sera for the identification of antigens present in GPIC-infected tissue culture cells but absent in the infectious elementary body (EB). Immunoblots of lysates of infected HeLa cells probed with the convalescent-phase sera identified protein antigens of 22, 34, and 52 kDa (p22, p34, and p52, respectively) that were not detected in lysates of purified EB or in uninfected HeLa cells. Protein p22 was also not detected in lysates of purified reticulate bodies. Immunoblotting of lysates of HeLa cells infected with other chlamydiae demonstrated that the antigenicity of p22 and p34 was subspecies specific. Immunoblotting was also used to detect p22 and p34 in lysates of the conjunctivae of infected guinea pigs. Adsorption of convalescent-phase sera with GPIC EB produced a reagent with dominant reactivity toward p22, p34, and a 28-kDa EB protein. Immunofluorescent staining of GPIC-infected HeLa cells demonstrated that these adsorbed sera labeled the inclusion and inclusion membrane, with no apparent reactivity toward EB or reticulate bodies. Collectively, these data identify non-EB chlamydial components which may be released into the inclusion during intracellular growth.

Interesting sequence differences between the pilin gene inversion regions of Moraxella lacunata ATCC 17956 and Moraxella bovis Epp63

The bacterium Moraxella lacunata is a causative agent of human conjunctivitis and keratitis. We have previously reported construction of plasmid pMxL1, which includes a 5.9-kb fragment on which the pilin gene inversion region of M. lacunata resides. The inversion region of pMxL1 was shown to invert when pMxL1 was in an Escherichia coli host cell. In this report, we present Western immunoblot analysis using Moraxella bovis Epp63 anti-I and anti-Q pilin sera which demonstrate that pMxL1 makes pilin only when in orientation 1. The sequence of the pMxL1 plasmid containing the invertible region contains a perfect tandem repeat of 19 bp in the orientation 1 nonexpressed pilin gene at the middle of the recombination junction site. This 19-bp insert causes a frameshift and disrupts the pilin gene. The predicted amino acid sequence of this nonfunctional pilin gene (with the 19-bp repeat subtracted) bears closest resemblance to M. bovis Epp63 Q pilin sequence, although the other (functional) M. lacunata pilin encoded by pMxL1 shows slightly higher homology to Q pilin. Comparison of the pMxL1 sequence with that of the M. bovis Epp63 sequence shows two other particularly interesting differences. One is a 15-bp sequence addition found in pMxL1 at the 60-bp region previously reported as a possible M. bovis recombinational enhancer. The second is an AT deletion in pMxL1 compared with Epp63 within an open reading frame (tfpB) which results in the pMxL1 tfpB open reading frame being one-third shorter than in Epp63. The DNA sequences in these three altered regions from the M. lacunata strain from which pMxL1 was derived were amplified by polymerase chain reaction and sequenced. The parent strain was found to contain the differences seen in pMxL1. Comparison of the M.bovis and M. lacunata pilin gene amino acid sequences is also presented.

Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata

Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.