Investigation of the pathogens contributing to naturally occurring outbreaks of infectious bovine keratoconjunctivitis (pinkeye) using Next Generation Sequencing.

Infectious bovine keratoconjunctivitis (IBK), commonly known as pinkeye, has a marked negative impact on the economy of the cattle industry. Moraxella species, including Mor. bovis and Mor. bovoculi, which have been associated with this disease, colonize clinically healthy eyes as well, suggesting that there are intrinsic changes that may occur to the ocular microbiota or the involvement of additional unrecognized organisms that contribute to IBK. To evaluate this, 104 ocular swabs collected from eyes with IBK or clinically healthy eyes from 16 different cattle herds were subjected to 16 S rRNA gene PCR and next generation sequencing (NGS) analysis. Organisms detected were similar across the herds and there was no difference in the total number of bacterial groups detected among IBK cases and controls. However, the percentages of the different organisms detected varied between the two groups, including Moraxella spp., with more Moraxella spp. in eyes with IBK than controls. Further, using culture and whole genome NGS, a new species of Moraxella (suggested name Mor. oculobovii) was detected from the eyes of cattle from two farms. This strain is non-hemolytic on blood agar, is missing the RTX operon, and is likely a non-pathogenic strain of the bovine ocular microbiome. Alteration of the ocular microbiota composition may have a predisposing role, enhancing bacterial infection and the occurrence of clinical IBK. Future studies are required to evaluate if these changes are permanent or if there is a shift in the microbiome following recovery from the infection and how antibiotics might affect the microbiome.

Cytological and microbiological evaluation of conjunctiva in camels with and without conjunctivitis.

OBJECTIVE: To describe the cytological analysis of conjunctiva from normal camels and camels with bacterial conjunctivitis. ANIMALS STUDIED: This study was conducted on 7 normal camels and 15 camels affected with conjunctivitis. The affected camels had a history of conjunctivitis with signs including chemosis, blepharospasm, frequent blinking, and mild-to-moderate serous, mucoid, or purulent ocular discharge. PROCEDURES: Bacterial swabs were collected from the inferior conjunctival sac of the affected eye without topical anesthetics. Conjunctival smears were obtained from the conjunctival surface for cytological analysis. RESULTS: The cellular analysis of ocular smears revealed a higher percentage of basal cells, neutrophils, eosinophils, and macrophages in camels with conjunctivitis compared with normal camels. In contrast to this, smears from normal camels showed an increased percentage of superficial epithelial cells compared with affected camels. The microbiological assessment of conjunctival swabs collected from affected animals identified a bacterial growth of Staphylococcus aureus., Bacillus sp., Streptococcus sp., Enterococcus faecium., Staphylococcus sp., Corynebacterium sp., Coryne pseudotuberculosis., Saprophytica, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus vulagaris, and Pseudomonas aeruginosa. CONCLUSIONS: It was observed that bacterial conjunctivitis in camels was associated with increased percentages of basal epithelial cells, neutrophils, eosinophils, and macrophages compared with normal camels, while normal camels showed an increased percentage of superficial epithelial cells compared with affected camels.

Antibiotic perturbation of gut bacteria does not significantly alter host responses to ocular disease in a songbird species.

Bacterial communities in and on wild hosts are increasingly appreciated for their importance in host health. Through both direct and indirect interactions, bacteria lining vertebrate gut mucosa provide hosts protection against infectious pathogens, sometimes even in distal body regions through immune regulation. In house finches (Haemorhous mexicanus), the bacterial pathogen Mycoplasma gallisepticum (MG) causes conjunctivitis, with ocular inflammation mediated by pro- and anti-inflammatory cytokines and infection triggering MG-specific antibodies. Here, we tested the role of gut bacteria in host responses to MG by using oral antibiotics to perturb bacteria in the gut of captive house finches prior to experimental inoculation with MG. We found no clear support for an impact of gut bacterial disruption on conjunctival pathology, MG load, or plasma antibody levels. However, there was a non-significant trend for birds with intact gut communities to have greater conjunctival pathology, suggesting a possible impact of gut bacteria on pro-inflammatory cytokine stimulation. Using 16S bacterial rRNA amplicon sequencing, we found dramatic differences in cloacal bacterial community composition between captive, wild-caught house finches in our experiment and free-living finches from the same population, with lower bacterial richness and core communities composed of fewer genera in captive finches. We hypothesize that captivity may have affected the strength of results in this experiment, necessitating further study with this consideration. The abundance of anthropogenic impacts on wildlife and their bacterial communities, alongside the emergence and spread of infectious diseases, highlights the importance of studies addressing the role of commensal bacteria in health and disease, and the consequences of gut bacterial shifts on wild hosts.

Retrospective Review of Mycobacterial Conjunctivitis in Cockatiels (Nymphicus hollandicus).

The etiologic disease organism responsible for causing mycobacteriosis in avian species is an acid-fast gram-positive bacterium. This bacterium causes granulomatous disease in various internal organs, but in cockatiels (Nymphicus hollandicus) it has been commonly identified within the conjunctival tissues. Twenty-six cases of mycobacterial conjunctivitis in cockatiels were diagnosed through histopathologic assessment of diseased tissue samples, Fite acid-fast staining, and polymerase chain reaction in this retrospective study. Clinicians who saw these cases were contacted, and information was obtained regarding recommended treatment protocols prescribed for the patients, the Mycobacterium species identified, and case outcomes. All patients in this retrospective study had a biopsy performed on the affected conjunctival tissue, and because of the small size of the patients, this excisional biopsy removed the affected tissue in its entirety or significantly debulked the lesion. Of the 26 cases, 10 were lost to follow-up, 4 were euthanatized, 7 died, and 5 were alive at the time this information was submitted for publication.

House finches with high coccidia burdens experience more severe experimental Mycoplasma gallisepticum infections.

Parasites co-infecting hosts can interact directly and indirectly to affect parasite growth and disease manifestation. We examined potential interactions between two common parasites of house finches: the bacterium Mycoplasma gallisepticum that causes conjunctivitis and the intestinal coccidian parasite Isospora sp. We quantified coccidia burdens prior to and following experimental infection with M. gallisepticum, exploiting the birds' range of natural coccidia burdens. Birds with greater baseline coccidia burdens developed higher M. gallisepticum loads and longer lasting conjunctivitis following inoculation. However, experimental inoculation with M. gallisepticum did not appear to alter coccidia shedding. Our study suggests that differences in immunocompetence or condition may predispose some finches to more severe infections with both pathogens.

Conjunctival bacterial flora and antimicrobial susceptibility of captive and free-living sea turtles in Brazil.

PURPOSE: To describe the aerobic conjunctival bacterial flora of 3 especies of free-living and under human care sea turtles and determine its antimicrobial susceptibility in vitro. METHOD: Thirty-six sea turtles (72 eyes), juveniles and adults, 7 free-living Chelonia mydas and 8 Chelonia mydas, 4 Caretta caretta, 11 Eretmochelys imbricata, and 6 Lepidochelys olivacea under human care, were evaluated. Conjunctival cultures were collected for identification of aerobic bacteria and antimicrobial susceptibility testing for ciprofloxacin, chloramphenicol, gentamicin, neomycin, oxacillin, polymyxin B, tetracycline, and tobramycin using antibiotic disks. Bacterial strains showing no sensitivity to 4 or more antimicrobials were considered multiresistant to this panel. RESULTS: Bacterial growth was observed in 12/14 (85.71%) samples in the free-living sea turtles, and there was growth in 100% (58/58) of the samples from captive animals. There were 94 strains isolated and 15 species identified. There was a predominance of Gram-positive bacteria in free-living Chelonia mydas, most of which were Bacillus and Staphylococcus. The most commonly isolated Gram-negative species were enterobacteria for free-living and under human care animals. The strains were predominantly sensitive to ciprofloxacin and tobramycin, and less sensitive to oxacillin or polymyxin B. Ten multiresistant strains were isolated. Yeast were identified in 13.89% (10/72) of the samples. CONCLUSIONS: These results, showing differences in the conjunctival bacterial flora of free-living and captive animals, may be helpful for diagnosis and treatment of ocular disorders in sea turtles.

Mycoplasmosis of House Finches ( Haemorhous mexicanus) and California Scrub-Jays ( Aphelocoma californica) in a Wildlife Rehabilitation Facility with Probable Nosocomial Transmission.

We describe an investigation of an outbreak of conjunctivitis in juvenile House Finches ( Haemorhous mexicanus) and California Scrub-jays ( Aphelocoma californica) at a central California, US wildlife rehabilitation facility. In late May 2015, the facility began admitting juvenile finches, the majority with normal eyes at intake. In June, with juvenile finches already present, the facility admitted juvenile scrub-jays, all with normal eyes at intake. In July, after conjunctivitis was observed in increasing numbers of juvenile finches and scrub-jays, carcasses were submitted for postmortem examination. Histopathology of five finches and three scrub-jays identified lymphocytic infiltrates in the ocular tissues. Conjunctival swabs from 87% (13/15) finches and 33% (4/12) scrub-jays were PCR-positive for Mycoplasma gallisepticum. One finch and two scrub-jays were PCR-positive for Mycoplasma synoviae. Additionally, gene sequencing (16S ribosomal RNA and 16S-23S intergenic spacer region) identified Mycoplasma sturni from 33% (3/9) scrub-jays. This outbreak of conjunctivitis suggested that M. gallisepticum-infected juvenile finches admitted to and maintained in a multispecies nursery likely resulted in transmission within the facility to healthy juvenile finches and scrub-jays. Evidence of other Mycoplasma spp. in finches and scrub-jays indicates that these species are susceptible to infection and may act as carriers. This outbreak highlighted the need for effective triage and biosecurity measures within wildlife rehabilitation facilities.

Determination of baseline values for routine ophthalmic tests in bearded capuchin (Sapajus libidinosus).

BACKGROUND: Establish baseline values for ophthalmic diagnostic tests in Sapajus libidinosus. METHODS: Ophthalmic diagnostic tests, namely Schirmer tear test 1 (STT-1), intraocular pressure (IOP), B-mode ultrasound, culture of the bacterial conjunctival microbiota, and conjunctival exfoliative cytology, were performed in 15 S. libidinosus. RESULTS: Mean values found were as follows: 2.50 ± 2.94 mm/min for the STT-1; 13.3 ± 3.32 mm Hg for the IOP; 2.47 ± 0.41 mm for the depth of the anterior chamber; 2.86 ± 0.96 mm for the axial length of the lens; 10.97 ± 0.48 mm for the depth of the vitreous chamber; and 16.32 ± 1.24 mm for the axial length of the eyeball. The bacterial genus most frequently found was Staphylococcus spp. Conjunctival cytology showed intermediate epithelial, squamous superficial epithelial, and keratinized cells. CONCLUSIONS: Determination of baseline values for eye measurements and ophthalmic tests will assist in the diagnosis of eye diseases in S. libidinosus monkeys.

Severe Conjunctivitis Associated with Chlamydia felis Infection in a Free-ranging Eurasian Lynx ( Lynx lynx).

A free-ranging adult Eurasian lynx ( Lynx lynx) captured in Switzerland presented with a severe purulent unilateral conjunctivitis. Chlamydia felis was detected in conjunctival swabs by real-time quantitative PCR. Systemic treatment with oxytetracycline and ketoprofen led to complete recovery. Infection with C. felis has not been previously reported in Eurasian lynx.

Mycobacterium genavense Infection in a Domestic Ferret (Mustela putorius furo).

Mycobacterium genavense infection was diagnosed in an adult ferret with ptosis of the left eye, a proliferative lesion of the conjunctiva of the nictitating membrane, conjunctival swelling, and tumefaction of the periorbital tissues with a watery ocular discharge and the presence of a retrobulbar mass. The diagnosis was based on characteristic cytology of the retrobulbar mass and left mandibular lymph node that revealed granulomatous inflammation. Ziehl-Neelsen staining showed the presence of positive acid-fast bacilli in the cytoplasm of the macrophages. The diagnosis was confirmed by sequence analysis of the 16S rRNA gene amplified by using a multiplex polymerase chain reaction from a fresh lymph node biopsy. Therapy with marbofloxacin, rifampicin, and clarithromycin was recommended for 6 months and after this period, the veterinarian who was treating the ferret reported the disappearance of clinical signs. Six months after the end of the antibiotic treatment, the symptoms described previously reoccurred. Confirmatory laboratory tests were not performed but a recurrence of M genavense infection was suspected and the veterinarian, in agreement with the owner, euthanized the ferret.

Characterization of unilateral conjunctival inoculation with Mycoplasma gallisepticum in house finches.

House finches in much of the continental United States experience annual epidemics of mycoplasmal conjunctivitis, caused by the bacterial pathogen Mycoplasma gallisepticum (MG). Although evidence suggests that natural infections typically begin unilaterally, experimental inoculations of songbirds with MG to date have all been administered bilaterally. Furthermore, studies of free-living finches find more severe clinical signs of mycoplasmal conjunctivitis in left versus right eyes, but the mechanisms underlying this side bias remain unknown. Here, we characterized unilateral inoculation of house finches with MG, and tested whether differential susceptibility of left versus right conjunctiva explains the side bias in disease severity of free-living finches. We directly inoculated house finches in either the left or right conjunctiva and characterized resulting disease severity and pathogen load throughout the course of infection. As expected, unilateral inoculation resulted in significantly more severe conjunctivitis, as well as higher conjunctival bacterial loads, on whichever side (left or right) birds were directly inoculated. However, in 55% of cases, unilateral inoculations resulted in bilateral disease, and in 85% cases there was evidence of bilateral infection. The overall severity of disease did not differ for birds inoculated in the left versus right conjunctiva, suggesting that physiological differences between the conjunctivae cannot explain the side bias in disease severity of free-living birds. Instead, laterality in exposure, perhaps due to feeding handedness, likely explains the detected field patterns. RESEARCH HIGHLIGHTS House finches show more severe disease in the directly inoculated conjunctiva. Unilateral inoculations lead to high rates of bilateral infection and disease. Overall disease severity does not differ for the left- or right-inoculated conjunctiva. Laterality in exposure likely explains the left-side bias in natural infections.

Sudden death of an Indian peafowl (Pavo cristatus) at a zoo due to non-pigmented Serratia marcescens infection.

A 16-year-old female Indian peafowl (Pavo cristatus) died two days after recognition of conjunctivitis in the right eye, anorexia and depression. Gross necropsy revealed a thick pseudomembrane under the eyelid and hydropericardium. Histopathological examination revealed hepatocellular necrosis, sinusoidal and vascular congestion and infiltrated inflammatory cells. Infiltration by inflammatory cells was noted in the epicardium. The lungs had mild interstitial pneumonia with the extensive congestion within the capillaries of the air sacs. Tubular interstitial congestion and necrosis was noted in the kidneys. Bacterial culture and nucleotide sequencing of the inflammatory specimens identified the causative agent as Serratia marcescens, an uncommon bacterium in birds. In summary, this study describes the sudden death of an Indian peafowl due to S. marcescens infection, which is rarely seen in animals.

Microbial community sequencing analysis of the calf eye microbiota and relationship to infectious bovine keratoconjunctivitis.

Infectious bovine keratoconjunctivitis (IBK) is an important production limiting disease in cattle. Moraxella bovis has historically been considered the primary causal agent; however, vaccines have not been consistently shown as effective in controlling disease incidence. The purpose of this study was to examine the bacterial community of calf eyes prior to disease onset using high-throughput sequencing of 16S ribosomal RNA and determine if it was associated with IBK occurrence. The study was designed as a case-control nested within a randomized controlled trial (RCT). Eye swabs were collected from all spring-born calves without clinical signs of IBK (t0 swabs) on a research farm with a previous history of IBK disease outbreaks. At follow-up or weaning, calves were diagnosed as IBK positive or negative. The lag time between enrollment swabs (t0) and IBK diagnosis ranged from approximately one to three months. Cases were randomly selected from IBK positive calves and controls were selected from IBK negative calves (i.e. calves that did not exhibit clinical signs of IBK throughout the course of the RCT). Analysis of the fold-change differences between cases and controls did not reveal large-scale distinctions in bacterial composition. However, principal component analysis suggested bacterial composition differences between calf management groups, which were based on dam parity. Moraxella was found to be among the top ten most abundant genera in our population; however, the difference in abundance was not significant between the cases and controls. No large-scale differences in the bacterial communities of calves that did or did not develop IBK were observed in our population. Nevertheless, it remains unclear whether the "natural" bacterial population of the calf might ultimately impact disease status. Further study is warranted to examine bacterial taxa that were observed to be significantly more abundant in the cases or controls as potential vaccines/therapeutic targets.

Analysis of IMP-1 type metallo-ß-lactamase-producing Acinetobacter radioresistens isolated from companion animals.

IMP-1 type metallo-ß-lactamase-producing (MBL-producing) Acinetobacter radioresistens was isolated from a dog with cystitis and a cat with conjunctivitis. The MBL-producing A. radioresistens isolates were resistant to all of the ß-lactam antibiotics used in the sensitivity tests, but were susceptible to gentamicin, amikacin, and minocycline. Also, one of the two strains of A. radioresistens was susceptible to ciprofloxacin and levofloxacin. These two cases were cured by administration of tetracyclines and fluoroquinolones, which elicited a positive result in the sensitivity tests. This report of the isolation of MBL-producing A. radioresistens in companion animals is the first in the world. To prevent the proliferation of MBL-producing bacteria, veterinary hospitals need to be aware of the behavior of MBL-producing organisms.

Epidemiology of bacterial conjunctivitis in chinchillas (Chinchilla lanigera): 49 cases (2005 to 2015).

OBJECTIVES: To evaluate the anamnesis, clinical signs, diagnostic test results, treatment and outcome of chinchillas diagnosed with bacterial conjunctivitis. METHODS: Medical records of 49 chinchillas diagnosed with bacterial conjunctivitis were retrospectively reviewed. Association between clinical signs and type of bacteria involved was determined by means of univariate logistic regression. RESULTS: 61·5% of the isolated bacteria were Gram-negative, and the most common bacterial species was Pseudomonas aeruginosa (50%), followed by Staphylococcus species (26·9%). Chinchillas with acute conjunctivitis (1 to 3 days) were much more commonly affected by Gram-negative organisms. The majority of chinchillas that presented with concurrent respiratory signs were diagnosed with P. aeruginosa. Clinical resolution of conjunctivitis was reported in 87·8% chinchillas with a median time to clinical resolution of 17·5 days. Susceptibility of P. aeruginosa isolates to potentiated sulphonamides, enrofloxacin, ciprofloxacin, gentamicin, amikacin and polymyxin B was 8·3, 36, 62·5, 88·5, 100 and 100%, respectively. CLINICAL SIGNIFICANCE: P. aeruginosa is the predominant bacterial species associated with bacterial conjunctivitis in chinchillas. With the exception of duration of clinical signs, information on the anamnesis or physical examination findings cannot aid in distinguishing conjunctivitis caused by P. aeruginosa or other Gram-negative bacteria from the ones caused by Gram-positive bacteria. Gentamicin- or polymyxin B-containing antibiotic formulations are recommended for empirical topical therapy.

Eye of the Finch: characterization of the ocular microbiome of house finches in relation to mycoplasmal conjunctivitis.

Vertebrate ocular microbiomes are poorly characterized and virtually unexplored in wildlife species. Pathogen defense is considered a key function of microbiomes, but determining microbiome stability during disease is critical for understanding the role of resident microbial communities in infectious disease dynamics. Here, we characterize the ocular bacterial microbiome of house finches (Haemorhous mexicanus), prior to and during experimental infection with an inflammatory ocular disease, Mycoplasmal conjunctivitis, caused by Mycoplasma gallisepticum. In ocular tissues of healthy house finches, we identified 526 total bacterial operational taxonomic units (OTUs, 97% similarity), primarily from Firmicutes (92.6%) and Proteobacteria (6.9%), via 16S rRNA gene amplicon sequencing. Resident ocular communities of healthy female finches were characterized by greater evenness and phylogenetic diversity compared to healthy male finches. Regardless of sex, ocular microbiome community structure significantly shifted 11 days after experimental inoculation with M. gallisepticum. A suite of OTUs, including taxa from the genera Methylobacterium, Acinetobacter and Mycoplasma, appear to drive these changes, indicating that the whole finch ocular microbiome responds to infection. Further study is needed to quantify changes in absolute abundance of resident taxa and to elucidate potential functional roles of the resident ocular microbiome in mediating individual responses to this common songbird bacterial pathogen.

Efficacy of Bdellovibrio bacteriovorus 109J for the treatment of dairy calves with experimentally induced infectious bovine keratoconjunctivitis.

OBJECTIVE To determine the efficacy of Bdellovibrio bacteriovorus 109J for the treatment of calves with experimentally induced infectious bovine keratoconjunctivitis (IBK). ANIMALS 12 healthy dairy calves. PROCEDURES For each calf, a grid keratotomy was performed on both eyes immediately before inoculation with Moraxella bovis hemolytic strain Epp63-300 (n = 11 calves) or nonhemolytic strain 12040577 (1 calf). For each calf inoculated with M bovis Epp63-300, the eyes were randomly assigned to receive an artificial tear solution with (treatment group) or without (control group) lyophilized B bacteriovorus 109J. Six doses of the assigned treatment (0.2 mL/eye, topically, q 48 h) were administered to each eye. On nontreatment days, eyes were assessed and corneal swab specimens and tear samples were collected for bacterial culture. Calves were euthanized 12 days after M bovis inoculation. The eyes were harvested for gross and histologic evaluation and bacterial culture. RESULTS The calf inoculated with M bovis 12040577 did not develop corneal ulcers. Of the 22 eyes inoculated with M bovis Epp63-300, 18 developed corneal ulcers consistent with IBK within 48 hours after inoculation; 4 of those eyes developed secondary corneal ulcers that were not consistent with IBK. Corneal ulcer size and severity and the time required for ulcer healing did not differ between the treatment and control groups. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that B bacteriovorus 109J was not effective for the treatment of IBK; however, the experimental model used produced lesions that did not completely mimic naturally occurring IBK.

House Finch (Haemorhous mexicanus) Conjunctivitis, and Mycoplasma spp. Isolated from North American Wild Birds, 1994-2015.

Sampling wild birds for mycoplasma culture has been key to the study of House Finch (Haemorhous mexicanus) conjunctivitis, yielding isolates of Mycoplasma gallisepticum spanning the temporal and geographic ranges of disease from emergence to endemicity. Faced with the challenges and costs of sample collection over time and from remote locations for submission to our laboratory for mycoplasma culture, protocols evolved to achieve a practical optimum. Herein we report making M. gallisepticum isolates from House Finches almost every year since the disease emerged in 1994, and we now have 227 isolates from 17 states. Our wild bird host range for M. gallisepticum isolates includes Blue Jay ( Cyanocitta cristata ), American Goldfinch (Spinus tristis), Lesser Goldfinch (Spinus psaltria), Purple Finch (Haemorhous purpureus), Evening Grosbeak ( Coccothraustes vespertinus ), and herein first reports for Western Scrub-jay ( Aphelocoma californica ), and American Crow ( Corvus brachyrhynchos ). By collecting and identifying isolates from birds with clinical signs similar to those of House Finch conjunctivitis, we also expanded the known host range of Mycoplasma sturni and obtained isolates from additional wild bird species. Accumulating evidence shows that a diverse range of wild bird species may carry or have been exposed to M. gallisepticum in the US, as in Europe and Asia. Therefore, the emergence of a pathogenic M. gallisepticum strain in House Finches may actually be the exception that has allowed us to identify the broader epidemiologic picture.

Feeder use predicts both acquisition and transmission of a contagious pathogen in a North American songbird.

Individual heterogeneity can influence the dynamics of infectious diseases in wildlife and humans alike. Thus, recent work has sought to identify behavioural characteristics that contribute disproportionately to individual variation in pathogen acquisition (super-receiving) or transmission (super-spreading). However, it remains unknown whether the same behaviours enhance both acquisition and transmission, a scenario likely to result in explosive epidemics. Here, we examined this possibility in an ecologically relevant host-pathogen system: house finches and their bacterial pathogen, Mycoplasma gallisepticum, which causes severe conjunctivitis. We examined behaviours likely to influence disease acquisition (feeder use, aggression, social network affiliations) in an observational field study, finding that the time an individual spends on bird feeders best predicted the risk of conjunctivitis. To test whether this behaviour also influences the likelihood of transmitting M. gallisepticum, we experimentally inoculated individuals based on feeding behaviour and tracked epidemics within captive flocks. As predicted, transmission was fastest when birds that spent the most time on feeders initiated the epidemic. Our results suggest that the same behaviour underlies both pathogen acquisition and transmission in this system and potentially others. Identifying individuals that exhibit such behaviours is critical for disease management.