the herd comprised approximately 550 dairy cows of the israeli - holstein breed raised in a zero - grazing management system .
heifers were transferred at the age of 2 months to another farm and returned pregnant 14 months later .
all the cows and heifers were observed at least twice daily by the herd s personnel and at least twice weekly by the attending veterinarian . cases of bnvv were observed during january through march 2002 .
clinical signs of bnvv developed in 32 of the 39 heifers . other age groups and heifers before calving were not affected .
treatment consisted of vaginal rinses with potassium permanganate or h2o2 daily from calving for as long as clinical symptoms persisted ( 9 ) .
local antimicrobial therapy was not attempted since it was ineffective during the outbreaks on the other farms .
a penicillin / streptomycin combination was given to 12 cows with temperatures > 39.5c , to provide broad - spectrum protection and prevent sepsis and limit economic losses ( metronidazole is the drug of choice to treat infections caused by anaerobic bacteria , but its use is prohibited in israel ) .
five of these cows developed metritis and peritonitis and had to be slaughtered , whereas the temperature of the remaining seven cows returned to normal .
no difference between convalescence periods of cows treated with potassium permanganate or h2o2 was observed .
eighteen cows recovered within 4 weeks after onset of infection ; in 9 , a more chronic infection , characterized by a mucopurulent vaginal discharge lasting up to 10 weeks , developed .
the swabs were kept in amies transport medium ( copan , italy ) and processed within 5 hours from sampling .
swabs for mycoplasma and ureaplasma were taken in mycoplasmal transport medium and hayflick s medium , respectively ( 10 ) .
swabs for virologic examination were placed in eagle s medium supplemented with antimicrobials and fetal calf serum and chilled .
after vortexing , the medium was used for polymerase chain reaction ( pcr ) assays and virus isolation .
vaginal biopsies were suspended directly in 10% formalin and stained with hematoxylin - eosin in the laboratory .
bacteriologic examination included aerobic , microaerophilic , and anaerobic cultures , and resulting microorganisms were identified by standard methods ( 10 ) .
samples were examined for chlamydia by direct immunofluorescence ( cellab , australia ) and for coxiella burnetti by the stamp ( 11 ) staining method .
the only microorganisms cultured consistently from affected cows but not from healthy ones were pigmented , gram - negative , non  spore - forming , anaerobic rods .
the number of pigmented colonies was directly related to the severity of the vulvovaginal lesions , and they were not cultured from cows after their recovery .
the pigmented bacteria were weakly saccharolytic ; negative for indole , catalase , esculin hydrolysis , -fucosidase , and -galactosidase ; and positive for -galactosidase and n - acetyl--glucosaminidase positive ( as determined by the api rapid i d 32a kit , [ biomrieux , france ] ) .
identification of pigmented isolates as p. levii was done according to the manual of clinical microbiology ( 12 ) . since susceptibility to vancomycin distinguishes porphyromonas spp . from other gram - negative anaerobic rods
( 12 ) the mic of the isolates to this antibiotic was determined by the etest method ( ab biodisk , sweden ) and was found to be 3 g / ml , indicating susceptibility . other potentially pathogenic bacteria
isolated occasionally included arcanobacterium pyogenes , -hemolytic streptococci , and several enterobacteriaceae , but they could not be correlated with either the clinical signs or the presence of p. levii or bovine herpesvirus 4 ( bohv-4 ) .
for the virologic examination , samples were added to confluent monolayers of madin - darby bovine kidney cells , observed daily for cytopathic effect , and passaged every 7 days .
bohv-4 was detected in samples from 27 cows with bnvv and from two cows in which the syndrome did not develop .
association of the bacteriologic or virologic findings and the clinical status of the cows was assessed statistically by the mantel - haenszel test ( statistix , analytical sotware , tallahassee , fl ) .
the test indicated a significant association between cows with bnvv and the presence of p. levii , adjusted for bohv-4 ( p = 0.0006 ) .
no statistically significant association between bohv-4 , adjusted for p. levii , and bnvv was found ( p = 0.2359 ) .
previous publications ( 13 ) also reported difficulties establishing a clear relationship between the presence of bohv-4 and , among other syndromes , bovine metritis , vaginitis , and abortions .
uterine biopsies were not taken , as injuring the mucosa in the presence of vaginal lesions could increase the risk for metritis .
bacterial colonies were seen in the section of vaginal biopsies , but typical changes of bohv-4 infection ( 13 ) were absent .
the single necropsied cow showed peritonitis , resulting from rectal rupture , the cause of which could not be determined .
the lesions affected the vulva and caudal part of the vagina but not the uterus ( figure 2 ) .
histopathologic examination of the vaginal lesions showed extensive necrosis of the epithelium and severe infiltration with a large number of mostly degenerative neutrophils and foamy macrophages . in the submucosa ,
since p. levii was isolated from all bnvv cases and very few healthy cows , it likely caused the lesions .
all the cases observed during this outbreak occurred in primiparous cows in a restricted geographic area ( within a radius of 20 km ) during a limited period of time , indicating that one or more risk factors , alone or in conjunction , predisposed cows to infection .
outbreaks were observed after a large number of cattle were introduced , affecting primarily the animals introduced into the host farm .
transportation and social conflict may have acted as stressors ( with stress immunosuppressive effects ) ( 14 ) and predisposed the introduced cows to infection , with only social conflict affecting the local cattle .
calving is a well - known stressor ( 15 ) , especially in primiparous cows ( 16 ) .
moreover , lesions of the genital tract caused by calving tend to be more severe in primiparous cows , thus making them more prone to infection .
after bnvv was described and its putative etiologic agent was identified , several sporadic cases were diagnosed on other farms , indicating that it might be underdiagnosed , and further studies of the syndrome may be warranted .
 an outbreak of bovine necrotic vulvovaginitis associated with porphyromonas levii , an emerging animal and human pathogen , affected 32 cows on a dairy farm in the northeast of israel . 
 five animals had to be culled . 
 this report appears to be the first that associates p. levii with bovine necrotic vulvovagnitis . 