Conjunctival cytological examination，bacteriological culture，and antimicrobial resistance profiles of healthy Mediterranean buffaloes（Bubalus bubalis）from Southern Italy

Barbara Lamagna，Maria Pia Pasolini，Sandra Nizza，Karina Mallardo，Maurizio Formicola，Alessandro Costagliola，Gerardo Fatone，Filomena Fiorito，Orlando Paciello，Luisa De Martino

Department of Veterinary Medicine and Animal Production，University of Naples Federico II，Via F.Delpino 1，80137 Naples，Italy

Conjunctival cytological examination，bacteriological culture，and antimicrobial resistance profiles of healthy Mediterranean buffaloes（Bubalus bubalis）from Southern Italy

Barbara Lamagna*，Maria Pia Pasolini，Sandra Nizza，Karina Mallardo，Maurizio Formicola，Alessandro Costagliola，Gerardo Fatone，Filomena Fiorito，Orlando Paciello，Luisa De Martino

Department of Veterinary Medicine and Animal Production，University of Naples Federico II，Via F.Delpino 1，80137 Naples，Italy

ARTICLE INFO

Article history：

Accepted 28 Jun 2015

Available online 18 Aug 2015

Mediterranean buffalo

Conjunctival cytological examination

Bacteriologic culture

Antimicrobial resistance

Objective：To assess normal conjunctival cytological and bacteriological/fungal flora features in the Mediterranean buffalo（Bubalus bubalis）.

Methods：Swabs were taken from the inferior conjunctival sac of both eyes of 57 healthy female buffaloes aged 24-36 months，with no evidence of ocular disease，farmed in Campania region（Southern Italy），for microbiological analysis.Conjunctival eye specimens of both eyes were subsequently obtained by a cyto-brush，for cytological analysis. The antimicrobial susceptibility of bacterial isolates was also determined using the diskdiffusion method on Mueller Hinton agar plates.

Results：Cytological examination of conjunctival swab specimens（114 eyes）revealed epithelial cells（basal，intermediate，columnar and superficial）in all samples，whereas neutrophils，lymphocytes and plasma cells were present in 70%，10%and 2%of samples，respectively.Microorganisms，for a total of 261 aerobic bacteria and 6 fungi，were isolated from 112/114 conjunctival samples［98.25%；95%confidence interval（CI）:93.18-99.70］.Only two conjunctival swabs did not yield bacteria and/or fungi（2/114，1.75%；95%CI:0.30-6.82）.Gram-positive aerobes were most commonly cultured（181/261，69.35%；95%CI:63.31-74.81），with Enterococcus faecium and Staphylococcus lentus predominating.Escherichia coli was the most frequently isolated as Gram-negative bacteria（80/261，30.65%；95%CI:25.19-36.69）.The antimicrobial resistance patterns of the isolated bacteria showed amoxycillin/clavulanic acid and cephalothin as the least sensitive antibiotics for both Gram-positive and Gram-negative bacteria.

Conclusions：These results provided first information on normal conjunctival ocular microflora and cytological features in Mediterranean buffalo.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.06.017

1.Introduction

Buffalo-breeding in Italy is an important zootechnical and economical reality，especially in Campania Region where buffalo herds represent 80%of the national buffalo assets.

Characterization of bacterial ocular flora of Mediterranean buffaloes is of special interest for animal and human health，since microorganisms can be transmitted to human populations that are in direct contact with these animals and can also be responsible for meat and milk deterioration.In addition，these animals have a potential role as vectors of antimicrobial resistant bacteria［1］.

To our knowledge，there are very few reports describing morphological and functional features of buffalo eye［2］，rare reports on congenital ocular abnormalities［3］，and no reports onconjunctivalmicrofloracytologicalfeaturesand/or incidence of ocular diseases in this species.Knowledge of the conjunctival flora of healthy buffaloes may be useful in the treatment of external ocular diseases.The aim of this studywas to evaluate cytologically and microbiologically the ocular conjunctiva of healthy Mediterranean buffaloes to determine the aerobic bacterial and fungal flora population and cytologic features of normal eyes.The antimicrobial susceptibility of bacterial isolates was also determined.

2.Materials and methods

2.1.Sample collection

Fifty-seven（114 eyes）clinically healthy female Mediterranean buffaloes，24-36 months old，were sampled in November 2014，in a farm in the province of Salerno（Campania Region，Italy）.Temperature was about 15°C and all Mediterranean buffaloes were housed outdoors in paddocks.

Each Mediterranean buffaloes underwent a thorough physical and ophthalmic examination including slit-lamp biomicroscopy and indirect ophthalmoscopy，to exclude animals with clinical signs of systemic and/or ocular diseases.The buffaloes were physically restrained by a skilled operator in a stock.The palpebral rim and the conjunctival sac of the lower lid were cleaned of mucus，debris and fluid.Pharmacologic induction of mydriasis was not necessary because of incomplete pupillary constriction.

For microbiology purpose，swabs were taken from the inferior conjunctival sac of both eyes of each buffalo.Topical anesthetics were not used.The microbiological sterile swab was removed aseptically from protective cover and was used dry until processing.The inferior eyelid was everted and specimens were collected by rotating the sterile swab.

Conjunctival eye specimens of both eyes were subsequently obtained by a cyto-brush，that was rotated in the inferior conjunctival fornix in a single direction five times and then lightly rolled onto a clean glass slide，as already described［4］. Slides were air-dried.

Swabs，in Amies transport medium（Oxoid Ltd，UK），and slides were sent to the laboratory on ice packs（4°C）within 4 h from collection.

2.2.Cytological examination

Slides were stained with May-Gr¨unwald-GiemsaQuik stain（Bio-Optica，Milan，Italy），observed with light microscope（E-600；Nikon Eclipse，Tokyo，Japan）at different magnifications and then examined at 40×magnification to differentiate and count the epithelial and inflammatory cells.The images were captured at the microscope coupled to a videocamera（DXM 1200F；Nikon Digital Camera，Tokyo，Japan），stored in the digital memory，and shown on the monitor.

Epithelial cells were classified as already described and quantified by counting 600 cells for each eye［5］.Inflammatory cells were counted for 20 high power field.The results were expressed as percentage of each epithelial cell and as median（range）of each inflammatory cell type.

2.3.Microbiological analysis

For conventional bacteriological detection，samples were cultured on blood agar base supplemented with 5%sheep blood，selective medium used for the isolation of Gram-positive microorganisms，on mannitol-salt agar，selective medium to identify staphylococci，and on MacConkey agar，selective and differential medium to grow Gram-negative bacteria.All agars were incubated for 24-48 h at 37°C under aerobic conditions. Colonies were then subjected to classic methods including Gram staining，colony morphology and biochemical tests for identification（Api System bioM`erieux），according to manufacturer's instructions.

The samples were also cultured for fungi on Sabouraud dextrose agar with chloramphenicol and incubated under normal atmospheric conditions at 30°C up to 7 days.The plates were all microbiological media from Oxoid Ltd，UK.

2.4.Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the disk-diffusion method on Mueller Hinton agar plates as recommended by the Clinical and Laboratory Standards Institute guidelines for veterinary isolates（CLSI 2008）.

Antimicrobialagentswere:amoxycillin/clavulanicacid（30μg），amikacin（30μg），bacitracin（10μg），chloramphenicol（30μg），ciprofloxacin（5μg），ceftazidime（30μg），clindamycin（2μg），ceftriaxone（30μg），cefuroxime（30μg），cefoperazone（30μg），doxycycline（30μg），ceftiofur（30μg），cephalothin（30μg），enrofloxacin（5μg），gentamicin（10μg），neomycin（30μg），sulphamethoxazole/trimethoprim（25μg），tetracycline（30μg），and tobramycin（30μg）.Antimicrobial disks were purchased from Oxoid Ltd，UK.For analysis purposes，isolates with intermediate susceptibility were recorded as resistant.

3.Results

3.1.Cytological findings

In all slides（100%）conjunctival cells identified as epithelial cells of various types（basal，intermediate，columnar and superficial）were observed（Figure 1）.Inclusion bodies were not noticed in any specimen.Basal（42%）and intermediate（40%）cells were the most represented，while columnar（11%）and superficial（7%）cells were rarely seen.

Neutrophils were observed in 80 eyes（70%）（median 3；range 0-30 cells）:in 34（30%）eyes they had a low incidence（1-5 cells），in 20 eyes（17%）they had a moderate incidence（5-10 cells）and in 26 eyes（23%）they had a high incidence（＞10）. In 25 eyes（22%）neutrophils were degenerate，with swollen pale nuclei（Figure 2）.

The lymphocytes were present in eleven eyes（10%）（0；0-18），always in association with neutrophils.Plasma cells were observed only in two eyes（2%）（0；0-3）.In 22 slides（19%）extracellular bacteria were found.In one slide a Malassezia spp. was identified.

3.2.Microbiological findings

From 114 conjunctival swabs，181 Gram-positive aerobic bacteria（181/261，69.35%；95%CI:63.31-74.81），and 80 Gram-negative aerobic bacteria（80/261，30.65%；95%CI: 25.19-36.69）were cultured（Table 1）.Only two conjunctival swabs did not yield bacteria（2/114，1.75%；95%CI:0.30-6.82）. All animals had a positive eye culture for one or both eyes. Moreover，we revealed 3/114（2.63%；95%CI:0.68-8.07）specimens as negative for Gram-positive bacteria culture，whereas 32/114（28.07%；95%CI:20.25-37.39）specimens as negative for Gram-negative bacteria culture.

Gram-positive were most commonly isolated with E.faecium and S.lentus predominating and showing the same percentage value（63/181，34.81%；95%CI:27.99-42.28）.The other isolates as Gram-positive bacteria were as follow:S.capitis（14），S.uberis（13），S.xylosus（12），G.sanguinis（9），A.viridans（1），S.hyicus（1），E.durans（1）and Micrococcus spp.（1）.E.coli was the most frequentlyisolatedasGram-negativebacteria（55/80，68.75%；95%CI:57.29-78.39）.Furthermore were identified E.cloacae（14 strains），E.sakazakii（4），C.koseri（2），Pantoea spp.4（1），Pantoea spp.3（1），Serratia odorigera（1），C.baumanii（1）and Raoutella ornithinolytica（1）.

Table 1 Microorganisms isolated from 114 conjunctival swabs of Mediterranean buffaloes.

Table 1（continued）

Table 1（continued）

Table 2 Resistance to nineteen antimicrobial agents among 181 Gram-positive isolates.

Table 3 Resistance to nineteen antimicrobial agents among 80 Gram-negative isolates.

Most conjunctival specimens were negative for growth of fungi.Fungal genera were recovered only in six specimens（6/ 114，5.26%；95%CI:2.16-11.57），and we identified Candida spp.（2），Alternaria spp.（3），and Malassezia spp.（1）.

Moreover，we observed 23 animals（23/57，40.35%；95%CI: 27.84-54.16）with bilateral positive eye culture presenting the sameconjunctivalbacteriological/fungalflorafeatureinbotheyes.

3.3.Antimicrobial susceptibility testing

The antibiotic sensitivity of isolated and identified Grampositive and Gram-negative are summarized in Tables 2 and 3，respectively.Amoxycillin/clavulanic acid，ceftazidime and cephalothin were the least sensitive antibiotics for Grampositive bacteria，whereas amoxycillin/clavulanic acid and cephalothin were the least sensitive antibiotics for Gramnegative bacteria.No Gram-negative bacteria showed enrofloxacin-resistance.

4.Discussion

Normal cytology findings of the eyes are rarely reported in the veterinary literature except for the canine，feline and equine conjunctival cells that are well described［4-6］.

The differential cell count of conjunctival epithelial cells，herein described，showed the highest mean densities in cylindrical and basal cells than intermediate and superficial cells.In contrast with our results in Mediterranean buffaloes，the average differential cell count in horses showed a majority of basal cells［6］，whereas a predominance of superficial cells was recorded in dogs［5］.

Furthermore，the presence of neutrophils and lymphocytes in 70%and 10%of samples，respectively in Mediterranean buffaloes differs from the data from normal eyes of cats and dogs，where the presence of lymphocytes and neutrophils resulted to be in low numbers［5］.

In animals that lack any clinical evidence of conjunctivitis，lymphocytes and plasma cells have been considered normal cytologic findings，while other leukocytes were supposed to be consistent with blood contamination，due to sampling［7］. However，in this study the high number of neutrophils and the highpercentageofdegenerateneutrophilswithout erythrocytes，observed in some slides，were consistent with a typical pattern of ocular surface inflammation，in absence of clinical signs.

In the present study only two conjunctival swabs did not yield bacteria.In cattle，a higher incidence（55%）of negative samples was reported in Turkey［8］，while bacteria were cultured from all tested eyes in North American bison［9］.Probably the recovery of bacteria in all conjunctival swabs from bison and Mediterranean buffaloes compared with cattle may be due，in part，to the different environmental conditions.These latter could play a critical role in conjunctival contamination.On the other hand，several parameters may influence the prevalence of certain microorganisms，such as geographical area，climate，season，species，hygienic conditions［10］.

Aerobic bacterial culture of conjunctival swab specimens in the present study yielded predominantly Gram-positive bacteria（69.35%）with Staphylococcus spp.and Enterococcus spp.most frequently isolated.Gram-negative bacteria were less commonly isolated（30.65%）.A significant presence of Gram-positive bacteria was recently reported in neotropical primates from Salvador，Brazil［11］.

In Turkey the most common bacteria isolated from the eyes of healthy cattle were Streptococcus and Staphylococcus spp.［8］. In USA，Hare et al.used molecular biology-based methods to determine the composition of bacterial flora in the conjunctivae of normal dairy and beef cattle from Maryland，and Staphylococcus crocelyticus，Staphylococcus sciuri and Gram-negative bacteriafromthefamilyEnterobacteriaceaeweremost commonly isolated［12］.In an Iranian study Bacillus cereus and Corynebacteriumpseudotubercolosispredominatedin conjunctival sac of normal cows［13］.Finally，conjunctival bacteriologic culture results from a herd of North American bison identified Bacillus spp.and Micrococcus spp.as the prevalent organisms in bison［9］.

However，Staphylococcus spp.show up as the most common Gram-positive bacteria isolated in our study（90/181，49.72%；95%CI:42.25-57.21%），as already reported in many mammalian species［10］.Staphylococcus are ubiquitous microflora of the skin and mucous membranes and they are common etiologic agent of suppurative ocular and periocular infections［10］.We identifiedasGram-positivebacteriaasinglestrainof S.hyicus，which role as commensal on the skin and mucosal surfaces is well known.In any case，also S.hyicus is a potential pathogen，characterized by positive reactions to the coagulase test，and may cause local lesions and serious opportunistic infections in animals and humans［14］.Of course，commensals can become pathogens in the right setting，such as debilitated or immunocompromised patients，or when the resident microbiota is altered by physical or chemical injuries of the eye.

The environmental conditions and behaviours of the buffaloes may explain the recovery of Enterococcus spp.and many bacteria of Enterobacteriaceae family（in particular genera: Escherichia and Enterobacter）from the conjunctivae of these animals.In many farms in Campania，buffaloes are kept in paddocks，in humid environments，all year long，and they have straight contact with their feces.E.faecium can be a commensal in the human intestine，but it may be also a pathogen，causing diseases like neonatal meningitis and sepsis［15，16］.

Furthermore，we found a relatively high presence of Gramnegative bacteria（80/261，30.65%；95%CI:25.19-36.69）with high prevalence of E.coli（55/80，68.75%；95%CI:57.29-78.39）that in the Mediterranean buffaloes can be attributed to their habitats.

Numerous fungi infect the eye either by direct introduction through trauma，by extension from infected adjacent tissues，or by hematogenous dissemination to the eye，but the percentage of these cases is usually low.Fungal infections of the eye are important amongst the clinical conditions responsible for ocular morbidity and blindness.Our specimens revealed the presence of fungal strains in the lowest percentage（6/114，5.26%；95%CI:2.16-11.57）.In Florida fungi were isolated from 100%of the healthy cows tested［17］.In Italy the total number of eyes of healthy cows positive for fungi ranged from 85%to 100%，from 65%to 95%，and from 55%to 95%on the basis of different management of the farms in which cows were housed［18］.

On the basis of the antimicrobial test，amoxycillin/clavulanic acid and cephalothin were the least sensitive antibiotics for both Gram-positive and Gram-negative bacteria.No Gram-negative bacteriashowedenrofloxacin-resistancewhichhasbeen already reported for E.coli isolated from dogs and cats［19］.

Monitoring antibiotic resistance patterns may serve as an early indicator of changes in antibiotic susceptibility of clinical isolates.The growing problem of antimicrobial resistance has become a significant public health concern worldwide，and it involves practically all types of bacteria both pathogenic and non pathogenic bacteria.

In conclusion，a different conjunctival flora in buffaloes in Campaniacomparedtopreviousreportsregardingcattleinvarious geographic area and North American bison was demonstrated；these differencesmaybecorrelatedwith environmental conditions and behaviours of the buffaloes；knowledge of normal ocular flora in buffaloes is very important in order to improve understanding aboutthecomparativeocularflora，tohelpanimmediatetherapyof externaloculardiseaseandtodiscernpathogensfromcontaminant.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

The authors thank the participating veterinarians and farmers for their constructive collaboration.This study was supported in part by the University of Naples“Federico II”，Fondi di Ateneo 2014.

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28 May 2015

Barbara Lamagna，University of Naples Federico II，Department of Veterinary Medicine and Animal Production，Via F.Delpino 1，80137 Naples，Italy.

Tel:+39 081 2536025

Fax:+39 081 2536021

E-mail:blamagna@unina.it

Peer review under responsibility of Hainan Medical University.

Foundation Project:Supported in part by the University of Naples“Federico II”，Fondi di Ateneo 2014.

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