mass deaths occurred at a large rearing facility for north american bullfrogs ( rana catesbeiana ) , 46 km from montevideo , uruguay .
the farm is a commercially managed operation , with trained employees under permanent , technical supervision .
mortality rates during the days before the outbreak were approximately 0.5% per week , which is considered normal for this type of farm .
frog growth rates were also within normal parameters for these conditions ( 10 ) . at this farm
, spawning occurs in 50-l tanks from october to february ( spring and summer ) , tadpoles grow until march , and metamorphosis occurs from march to may ( fall season ) .
newly metamorphosed frogs ( 58 g ) are harvested from tadpole ponds and stocked in tanks with circulating water until resorption of the tail is complete .
frogs are then transferred to large growing tanks , in which the frogs are reared at a density of 500 per square meter while standing in shallow water with only their bodies immersed ( 10 ) . permanently running water
frogs are fed with trout chow ( purina aquamax carnivorous ) and earthworms ( eisenia foetida ) cultured at the farm .
tanks are cleaned every day to eliminate excess feed and feces , and animals are sorted to maintain homogeneous sizes in each tank .
earlier in the same week , a tank containing 5,000 recently metamorphosed frogs had been emptied , and the population placed in three other tanks along with other frogs that had metamorphosed during the same week .
the following day , frogs started to die in unusually high numbers ( 300 deaths on day 1 ) in these three tanks . to reduce the risk of bacterial infections , poly - hexamethylene - biguanide - hydrochloride ( vantocil ib , zeneca biocides , blackley , manchester , uk )
the deaths continued and spread to other tanks ; 15,000 frogs died during the first week .
an antifungal therapy ( benzalkonium chloride 2 ppm baths ,  farmazul  1% solution , lab .
farmaco - uruguayo , acua de figueroa , montevideo ) ( 11 ) was used , but this treatment was also ineffective and almost 95% of the farm s total population of recently metamorphosed frogs died within 30 days .
mortality rates in individual tanks were extremely high , with affected tanks losing 10% of the population on day 1 and usually > 90% of the population by day 3 or 4 . by day 10 , all frogs in the three index tanks were dead , and the disease had spread to metamorphs in other tanks on the farm .
the epidemic continued for 26 days ( until may 27 ) , when only 2,000 of the original 30,000 metamorphs remained .
none of the broodstock was affected , even though these animals were in close spatial association with the affected tanks .
although nonspecific , these signs , the high mortality rate , and the age of affected frogs are consistent with those reported for chytridiomycosis ( 2 ) .
many of the frogs had agonal convulsions and extension of hind limbs ; death occurred 1224 h later .
reddening of ventral skin was found in some cases a few hours before death , usually in the pelvic and femoral areas .
routine bacteriologic tests ( culture on blood agar for 24 h and 48 h at 37c ) were performed on liver , kidney , and heart blood collected from a sample of euthanized frogs , but these tests did not implicate causative pathogens .
necropsies were performed systematically on 30 frogs , but no gross abnormalities of the viscera were found .
internal organs ( lung , liver , kidney , heart , and spleen ) from 10 of these frogs were examined by histologic testing , but no lesions were detected .
examination of skin smears ( wet - mounted , stained with a 1:1 mixture of cotton blue [ parker ink ] and 10% aq .
koh ) , and histologic and ultrastructural analyses of fixed skin indicated subspherical developing sporangia and flask - shaped mature sporangia within the superficial layer of the epidermis ( figure ) .
septa , characteristic of b. dendrobatidis , were observed in many developing sporangia ( figure ) .
slight hyperplasia of the keratinized cells of the stratum corneum and stratum granulosum was observed in some areas where chytrid developmental stages were present .
specific examinations for viruses were not performed , as facilities to test for viruses were unavailable .
characteristic sporangia ( s ) containing zoospores ( z ) are visible in the epidermis ( asterisk , superficial epidermis ; arrow , septum within an empty sporangium ; bars , 10 m .
c , skin smear from infected frog , stained with 1:1 cotton blue and 10% aqueous potassium hydroxide ( aq koh ) ( d , developing stages of batrachochytrium dendrobatidis ; arrow , septum within a sporangium ; bar , 10 m .
d , electron micrograph of an empty sporangium showing diagnostic septum ( arrow ) ( bar , 2 m ) . in a basic transmission experiment ,
two groups of five apparently healthy frogs from affected tanks were placed in separate aquaria , one contained five frogs from an unaffected farm in uruguay and the other empty .
deaths began on day 4 , with death occurring 2448 h after first signs of disease . within 8 days ,
after the incident , death rates returned to levels before the event . as a result of this event , monthly surveillance for cutaneous chytrid infection
was instigated ; ventral hind limbs , feet , and digits are examined from five healthy frogs and from any sick or dead frogs .
b. dendrobatidis has been repeatedly observed in skin smears of healthy frogs from the farm at which the outbreak occurred .
similar results have since been obtained from six other bullfrog farms in uruguay ( r. mazzoni , unpub . data ) .
histologic examination of infected frogs on which necropsies were performed since the 1999 epidemic shows focal mild hyperplasia of the superficial epidermis associated with sporangia of b. dendrobatidis .
cutaneous chytrid infection was detected during an investigation into mass deaths in farmed north american bullfrogs . although chytridiomycosis has been reported as an important cause of mass deaths in wild amphibians , whether chytrid infection was the cause of deaths in the disease outbreak described in this paper is unclear .
first , while no examinations for b. dendrobatidis had been performed before the deaths , this organism has been repeatedly observed in skin examinations of healthy frogs in follow - up studies .
second , although not proved efficacious against cutaneous chytridiomycosis , the use of benzalkonium chloride , a recognized therapy for infections with nonhyphal fungi , failed to reduce mortality rates .
possible causative agents include an infectious pathogen , which could not be detected by the examinations used , such as a ranavirus ( 2 ) or adverse environmental factors .
alternatively , chytridiomycosis could be the cause of the outbreak if the current chytrid is a different strain from that causing the outbreak or became attenuated , if the farmed bullfrogs had immunity to the pathogen , or if a change in the environment before the die - off allowed a normally benign infection to become pathogenic .
for example , increased temperatures reversibly inhibit both growth of b. dendrobatidis in culture ( 3 ) and the progress of disease outbreaks ( d. nichols , smithsonian institute , pers .
the deaths we describe occurred at the beginning of winter and may have been precipitated by lowered environmental temperatures .
the rearing of bullfrogs is a growth industry in south america , particularly brazil , uruguay , and argentina .
anecdotal reports of similar loss of stock in a number of farms in uruguay ( r. mazzoni , unpub .
data ) suggest that disease is a major economic threat to this industry in south america .
this situation is exacerbated by a lack of veterinary diagnostic capacity , management protocols , and treatment or prophylaxis .
regardless of the cause of the deaths , the north american bullfrog may be relatively resistant to chytridiomycosis , at least under the environmental conditions in the current study .
unpublished data suggest that metamorph bullfrogs can be experimentally infected by b. dendrobatidis without developing signs of disease ( 12 ) , and others have noted differential susceptibility to this disease among amphibian species ( 1,2 ) . nonclinical ( silent ) infections in farmed bullfrogs suggest that this species may act as a carrier of chytridiomycosis , which has serious implications for conservation of biodiversity .
b. dendrobatidis has been implicated in the complete removal of multiple species amphibian populations over large geographic areas in the wild ( 1,2 ) .
farmed bullfrogs originate in north america and have been introduced to south america during the last few decades for the lucrative restaurant trade .
live , farmed south american frogs are exported to other south american countries and to the united states .
the trade in bullfrogs is large in scale and global in scope . according to the u.s .
fish and wildlife service , over 1 million bullfrogs are imported into the united states each year from south america alone ( 9 ) , with others shipped in from asia . in the united states ,
recently , guidelines for amphibian translocations have been published that include specific quarantine and testing recommendations for chytridiomycosis ( 13 ) .
these procedures , however , are intended for animal movements for international union for conservation of nature and natural resources programs and are not legally binding . in the united states , the u.s .
department of agriculture ( usda ) may be the most suitable regulatory body for the bullfrog trade since these animals are a commercial agricultural product .
the farm we studied was opened in october 1998 with stock that originated from brazil .
the brazilian farmers originally imported 300 pairs of frogs from canada to set up the first south american farms approximately 30 years ago .
the pathogen has been present in bullfrogs in north america since at least the 1970s ( 14 ) .
b. dendrobatidis in this outbreak may have been imported with breeding stock from north america .
the uruguay farm in our study is enclosed by concrete walls , but outdoor ponds have open access to wildlife , and wild amphibians ( tree frogs ) have been observed in the greenhouses where indoor rearing tanks are located .
whatever the origins of b. dendrobatidis in farmed bullfrogs in uruguay , such open - plan farms are likely sources of infection and disease for wild amphibian species . in view of this risk ,
surveillance for chytrid infection and for unusual levels of deaths in endemic amphibian species should be established in the vicinity of frog farms in uruguay and elsewhere in the world .
global trade and commerce are regularly cited as drivers of disease emergence in humans , domestic animals , wildlife , and plants ( 6,1518 ) . for wildlife
emerging infectious diseases , quantitative analyses demonstrate that this process is the most important driving factor ( 8) . for example , the ( unknown ) causative agent of the bullfrog deaths we describe may be spread by such trade . also , the identification of b. dendrobatidis in an international food animal trade has implications for amphibian conservation and for disease emergence in general .
our study adds to reports of chytridiomycosis in the international pet trade ( 19 ) , amphibians for outdoor pond stocking in the united states ( 2,20 ) , importation for zoo collections ( 5 ) , trade in laboratory animals ( 21 ) , and species known to have recently been introduced into new geographic regions ( e.g. , cane toads in australia ) ( 1 ) . with a continued rise in the international air transport volume ( 22 ) , we predict a growing impact of trade and commerce on disease emergence within all populations , including wildlife .
for this reason , we urge a revision of national and international veterinary guidelines for the inspection and quarantine of imported animals and animal products .
 chytridiomycosis is an emerging disease responsible for global decline and extinction of amphibians . 
 we report the causative agent , batrachochytrium dendrobatidis , in north american bullfrogs ( rana catesbeiana ) farmed for the international restaurant trade . 
 our findings suggest that international trade may play a key role in the global dissemination of this and other emerging infectious diseases in wildlife . 
The Study
Conclusions