BRUCELLOSIS IN ELK. II. CLINICAL EFFECTS AND MEANS OF TRANSMISSION AS DETERMINED THROUGH ARTIFICIAL INFECTIONS

Transcription

1 BRUCELLOSIS IN ELK. II. CLINICAL EFFECTS AND MEANS OF TRANSMISSION AS DETERMINED THROUGH ARTIFICIAL INFECTIONS Authors: E. TOM THORNE, JAMIE K. MORTON, FLOYD M. BLUNT, and HUEY A. DAWSON Source: Journal of Wildlife Diseases, 14(3) : Published By: Wildlife Disease Association URL: BioOne Complete (complete.bioone.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at Usage of BioOne Complete content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

2 280 Journal of Wildlife Diseases Vol. 14, July, 1978 BRUCELLOSIS IN ELK. II. CLINICAL EFFECTS AND MEANS OF TRANSMISSION AS DETERMINED THROUGH ARTIFICIAL INFECTIONS E. TOM THORNE and JAMIE K. MORTON, Wyoming Game and Fish Department, Research Laboratory, Box 3312, University Station, Laramie, Wyoming 82071, USA. FLOYD M. BLUNT and HUEY A. DAWSON, Wyoming Game and Fish Department, Sybille Game Research Unit, Boiler Route, Wheatland, Wyoming 82201, USA. Abstract: The effects of brucebbosis in 60 mature elk (Cervus canadensis) and over 72 of their offspring were determined over a 65-month period. Artificial infections were induced with Brucella abortus type 1 strain All 27 artificially inoculated and 96% of 24 naturally-exposed mature elk became infected with brucelbosis. An additional five cow elk were used to examine the importance of venereal transmission. The average incubation period from artificial exposure until abortion was 89 days among seven cows, and the average incubation period from exposure to development of a serotiter was 39 days among 24 artificially-inoculated cows. The most probable route of infection was oral contact through licking or ingestion of contaminated materials. Fetal fluids, vaginal exudates and aborted fetuses were the most likely sources of contamination. The venereal route of transmission was unimportant. Abortion or birth of nonviable calves was the most important and frequent sign of brucellosis, and 48% of 29 cows lost their first calf following infection. Other signs were secondarily infected hygromata and synovitis in the lower legs. Most calves born alive to infected cows demonstrated a serologic titer at or soon after birth, and the majority lost their titer. Many of the calves demonstrating early postnatal titers as well as those born without indication of infection became infected later in life. Although the maximum duration of brucellosis was not determined, one cow had maintained an infection for 56 months prior to necropsy. Five elk apparently recovered, but Brucella was recovered at necropsy from another three which also appeared to be recovering. INTRODUCTION Brucellosis is an infectious disease of animals and man caused by members of the genus Brucella. Although it has been identified in many species of wildlife throughout the world,37 little effort has been made to determine the effects of brucellosis in native fauna. Brucellosis in wildlife in the United States was first diagnosed in 1917 in bison (Bison bison) in Yellowstone National Park Since then the disease frequently has been diagnosed in free-ranging bison in the United States and Canada. Scrotal enlargement and orchitis have been the primary signs described, and Brucella abort us has been recovered from several bulls.4 4 Abortions by bison were observed on winter feeding grounds in Elk Island National Park, Canada by Corner and Connell4 who felt that a low calf crop might have been attributable to brucellosis. Natural infections with brucellosis have occurred in moose (Alces alces) with two cases described in the United States6 #{176} and two cases in Canada.4 Brucellosis in moose differs from the disease in other animals by producing generalized, apparently fatal infection. In moose brucellosis has been characterized by emaciation, fibrinous

3 Journal of Wildlife Diseases Vol. 14, July, pleuritis, pericarditis and peritonitis, enlarged edematous lymph nodes and focal necrosis variously located in the liver, kidneys and spleen. The scrotum or testicles were involved in two bulls. B. abortus resembling that causing bovine brucellosis was recovered. The extremely rare occurrence of positive serologic reactions to brucellosis in moose may be due to its usually fatal termination. Rangiferine brucellosis is an enzootic disease of caribou (Rangifer tarandus) caused by B. suis biotype 4. The natural infection has been characterized by orchitis-epididymitis, bursitis-synovitis and metritis with abortion and retained placental membranes Infections in caribou-hunting peoples have resulted from contact with rangifers infected with brucellosis Youatt and Fay38 inoculated two 1.5- year old male white-tailed deer (Odocoileus uirginianus) with 10 million B. abortus type 1 strain Standard tube agglutination (STT) titers were first detected at 2 and 3 weeks with high titers of 1600 and 3200 being reached at 20 and 26 weeks, respectively, by the two deer. Titers remained high until conclusion of the experiment 20 weeks later. Baker et al. produced infections in two whitetailed does using strain Both does developed STT titers of 100 or greater within 42 days, but they did not go over 200. One maintained a high titer longer than the other, but both had declined to 25 by 555 days, at study termination. The doe with the lower titer did not produce fawns the first year after infection, but produced a small healthy fawn the second year. Small nonviable fawns were produced each year by the doe with the higher titer. All gestation periods were shortened. Attempts to isolate Brucella from the nonviable fawns and the doe with the higher titer at 5 days following parturition were unsuccessful. Neither of two control does nor a breeding buck developed significant titers. The authors decided the results, although inconclusive, raise some question regarding the effects of brucellosis on reproduction in white-tailed deer. Brucellosis was first detected in elk in Wyoming in the early 1930 s.22,14 More recently the disease has been shown to be limited to winter feedground herds of the western portion of the state, and B. abortus type 1 has been recovered from aborted fetuses and naturally-infected elk. Brucellosis has been present in at least two of these herds for a number of years, and the incidence among mature cows has been approximately 5Y for the last 6 years. 3 To determine the effects of brucellosis on elk, controlled studies were done at the Wyoming Game and Fish Department s Sybille Game Research Unit from July, 1971 to November, This paper reports the clinical effects of the disease as determined by artificial infection and the results of natural exposure to artificially infected elk. MATERIALS AND METHODS Facilities Elk holding facilities consisted of a series of pole corrals ranging from 15 mx 15 m to 23 mx 66 m and a woven wire pen of approximately 1 ha. The facilities prevented exposure of other game animals in the Sybille area and of domestic livestock on lands adjacent to the Unit. Depending on the numbers involved, elk often were allowed the use of several adjoining corrals or pens to provide as much space as possible for exercise and to seek privacy. At times the elk were divided into two similar groups to facilitate identification of fetuses and calves with their mothers. Shelters and heated water were available in all corrals, and good quality alfalfa hay, fed at a maintenance level, was provided once daily on the ground. Human disturbances and necessary handling procedures were conducted in a manner to minimize stress to the elk, and many of them became quite tame during the study. The available laboratory facilities permitted indoor necropsies and prompt initiation of bacteriologic procedures.

4 282 Journal of Wildlife Diseases Vol. 14, July, 1978 Elk Most elk were trapped wild on the National Elk Refuge, Jackson, Wyoming and transported to the research unit during the winter months. A few were from the resident Sybille Unit herd. An attempt was made to select young, sexually mature cows and bulls. Trapped elk were screened for Brucella antibodies at the trap site using the buffered Brucella antigen (BBA) rapid card test. Sera from blood samples collected at the trap were later tested by the standard plate agglutination (SPT), rivanol (Riv), complement fixation (CFT) and BBA tests in the laboratory. All elk were retested with negative results one to three times over a 2 to 8-week period before being used in the study. A total of 60 adult elk was introduced into the study, and 56 calves were born alive during the investigation. The elk were formed and held as one or two small herds which simulated, as nearly as possible, a natural situation in their yearly life cycle. The original herd was formed in February, 1972 and consisted of seven Brucella inoculated (at approximately 5 months of an 8.5-month gestation period) and two uninoculated mature cows. Seven artificially inoculated (at approximately 6.5 months gestation) and three uninoculated cows were added in March, All but one of these 19 elk were determined pregnant by rectal palpation. Between August, 1973 and October, 1975, 10 Brucella inoculated and 13 uninoculated cow elk and 6 uninoculated bull elk were added. A separate group consisting of three inoculated bulls and five uninoculated cows was formed in the summer of 1973 to determine if brucellosis might be transmitted venereally from infected bulls to susceptible cows. Periodically throughout the study, elk were examined at necropsy at various stages of infection for bacteriologic and pathologic studies. All elk were eventually examined at necropsy. Infective Dose of Brucella On 29 July, 1971 avirulentcultureofb. abortus type 1 strain 2308, which had recently been passed through guinea pigs, was placed in a saline suspension containing 109 colony forming units (cfu) per 1.0 ml at 95% transmission using a spectrophotometer.1 This suspension was further adjusted to produce two suspensions one containing approximately 7.5 X 106 cfu and one approximately 7.5 X 10 cfu per ml. Four mature cow elk were isolated, two were inoculated with 7.5 X 10 cfu in 0.1 ml saline, which approximates the standard U.S. Department of Agriculture (USDA) infective dose for bovines, 4 7 and two others were inoculated with 7.5 X 106 cfu. These, and all subsequent inoculations, were made via the conjunctival sac. Jugular blood samples were collected frequently for hemaculture and serology. A serologic response by the SPT was first detected in high dose elk 20 days postinoculation (P1) and 26 days P1 in low dose elk. Among the high dose elk the BBA, Riv and CFT tests produced reactions on 26, 20 and 26 days, respectively. Sera from the low dose elk reacted to the BBA, Riv and CFT tests on 40,40 and 32 days P1, respectively. Titers of high dose elk became higher faster and remained high while those of low dose elk did not rise as high and began to decline in 4 to 6 months. All four elk produced Brucella positive hemacultures at 32 days P1. Based on the rate of development and extent and duration of serologic titers, 7.5 X 106 cfu was chosen as the dose for all subsequent artificial infections which were induced using a virulent culture recently passed through guinea pigs. Serology Periodically, jugular blood samples for serologic testing were collected aseptical- W Spectronic 20, Bausch and Lamb, Rochester, New York 14602, USA.

5 Journal of Wildlife Diseases Vol. 14, July, ly from all elk. Blood samples were obtained from calves as soon as possible following birth. Standard procedures of the USDA, Animal and Plant Health Inspection Service, were used for conducting the SPT, BBA and Riv tests. 5 6 A CFT, patterned after that described by Jones et al. was used during 1971 through Beginning in 1974, the CFT was modified and patterned after the microtiter technique for the diagnosis of bovine anaplasmosis. 9 When serology alone was used to diagnose brucellosis in experimental elk, serum producing any degree of reaction to two or more tests was considered positive. If a sample reacted to only one of the four tests, standards established for nonvaccinated cattle were used for the SPT, BBA and Riv tests. Complete fixation at the 1:40 dilution or higher was considered positive on the CFT. Pathology Aborted fetuses and newborn calves dying soon after birth were removed only long enough to collect samples for pathologic and bacteriologic examinations, usually a period of mm. Abdominal and cutaneous incisions were closed using surgical gut and the fetuses or calves returned to the location where they were found. Since fetuses and calves were considered to be possible means of brucellosis transmission, they were left in the corrals for a minimum of 8 hand up to a maximum of 24 h following discovery. Fetal ages were determined after Taber. 2 Elk were killed by gunshot in the head. Thorough necropsies were performed, gross pathologic changes noted and appropriate tissues were fixed in 1(Y formaim for histopathologic study. Bacteriology Blood samples were collected from all elk, and hemacultures were frequently prepared to detect Brucella in circulating blood. Four to 6 ml of blood were inoculated into a bottle containing a trypticase soy agara slant overlaid with 20 ml of tryptose brothu with 1% sodium citrate. Hemacultures were checked for growth weekly for 5 weeks. Bacteriologic cultures for Brucella were prepared from the stomach, spleen, liver and lung of aborted fetuses and nonviable calves. Bacteriologic samples were taken from other locations, depending on the condition of the individual fetus or calf. Several semen samples were obtained by electroejaculation during the breeding season (September and October) and, when appropriate, vaginal swabs were prepared from postparturient cows for Brucella isolation. At necropsy, aseptic techniques were used to collect, depending on sex, the following tissues: mandibular, suprapharyngeal, atlanta!, prescapular, prefemoral, popliteal, external and internal iliac, mesenteric, gastrohepatic, ileocecal, mediastinal, bronchial, supramammary and superficial inguinal lymph glands; spleen, liver, biceps femoris muscle, uterus, udder, cervix, testes, epididymides, seminal vesicles and ampu!lae; and fetal fluids, stomach contents, lung, liver and spleen. Both of paired structures were collected. Immediately after collection, tissues were flamed, halved, the surface minced20 and duplicate plates of media were inoculated. Serum-enriched trypticase soy agar with 30 mg cycloheximide and 1:800,000 crystal violet added per liter was used for most isolation attempts. Grossly contaminated samples such as feces were also cultured on a second selective media consisting of Trypticase#{174} Soy Agar, Baltimore Biological Laboratory, Cockeyeville, Maryland 21030, USA. Tryptose Broth,#{174}Difco Laboratories, Detroit, Michigan 48233, USA. Actidione,#{174} the Upjohn Company, Kalamazoo, Michigan 49001, USA.

6 284 Journal of Wildlife Diseases Vol. 14, July, 1978 tryptose broth, 25 g; agaru,20 g; tergitol 7, 0.15 ml; Tween 40, 25 ml; ethyl violet, 1.4 mg; sodium lauryl sulfate, 1.44 g; CNV, 1 vial; cycloheximide, 500mg per liter (Deyoe, B. L Pers. comm.). One set of plates was incubated under 10% CO2 and the other under atmospheric conditions to differentiate the field strain of B. abortus type 1 which requires CO2 from research strain 2308 which grows under atmospheric conditions. Representative cultures were submitted for confirmation as B. abortus type 1 strain 2308 to Biologic Reagents Section, Veterinary Services, Diagnostic Laboratory, Ames, Iowa 50010, USA. RESULTS AND DISCUSSION Morbidity In addition to the four elk used to determine an optimum infective dose of B. abortus, all 24 mature cows and 3 mature bulls became infected as indicated by a serologic response following artificial exposure. All but three of these infections were bacteriologically confirmed by hemaculture and/or at necropsy. These same three cows produced relatively low serotiters which were intermittent or of short duration. Seventeen (94%) of 18 uninoculated mature and presumably susceptible cows introduced into the study became infected. All were confirmed bacteriologically. Six (100%) uninoculated bulls introduced at 24 to 36 months of age acquired the infection through natural means, but one was not confirmed bacteriologically. All three bulls in the group formed to examine venereal transmission also became infected following artificial exposure. Establishment of brucellosis in the bovine depends upon the resistance of the animal, its age, reproductive status and degree of exposure.2 2 Elk undoubtedly responded similarly. Close confinement possibly contributed to a higher rate of morbidity than would occur with free-ranging elk, but contact among these research elk was no closer than occurs among animals in some feedground herds in western Wyoming during winter and early spring months. Incubation Period The incubation period for brucellosis may be considered as the time from exposure until clinical manifestation, usually abortion, or the time from exposure until the appearance of circulating antibodies, such as a 100 SPT titer.23 In cattle the incubation period may vary from 14 to 251 days, and there is an inverse relationship between stage of pregnancy and incubation period. The dose of organisms received at exposure also influences the incubation period in cattle.23 3 Among the artificially infected elk in this study, the incubation time in terms of occurrence of abortion was from 68 to 125 (average 89) days in seven cows which aborted. The incubation time from the most likely natural exposure until abortion was 44 days in one susceptible mature cow for which this could be determined. Three other susceptible mature cows aborted or gave birth to nonviable calves following infection, but the source of infection could not be determined. The period from detection of initial serotiter until calf loss among these cows ranged from (average 310) days. Appearance of a positive antibody titer by our criteria for defining an infected animal occurred in 21 to 91 days (average 39 days) in 24 artificially infected cows. One-third of these cows produced reactor titers by 3 weeks and three-fourths by 5 weeks postinoculation. Serologic developments and evaluation of the serologic tests used will be published later. Two females apparently had latent infections which were not detected by Bacto-Agar,#{174} Difco Laboratories, Detroit, Michigan 48233, USA. Cholestimethate, nystatin, vancomycin,#{174} Difco Laboratories, Detroit, Michigan 48233, USA.

7 Journal of Wildlife Diseases Vol. 14, July, serology or hemaculture from birth to 23 months of age. At 23 months, serologic titers developed followed by abortion 3 weeks later. A male calf had a postnatal titer of 8 weeks duration and then remained sero-negative. Recovery of Brucella at necropsy in this 15-month old bull possibly indicated a latent infection. A recently acquired infection by these elk could not be ruled out, but there was no known source of transmission at that time. Latent undetected infections that become active at parturition have been reported in cattle.5 23 Routes of Infection and Excretion of Brucella Cunningham5 reported the oral route of infection was the only one of importance in bovine brucellosis and most cattle become infected through licking or ingestion of contaminated materials. The same was apparently true with elk in this study. In 47 (77%) of 61 Brucella positive elk, Brucella was isolated from the mandibular, suprapharyngeal or atlanta! lymph glands at necropsy. In five elk, one or more of these lymph glands were the only sites of isolation. These lymph glands primarily receive afferent vessels from the oral cavity and related structures, and their infection indicates the oral or conjunctival route of entry by the organism.5 28 Thirteen of the 21 naturally-acquired infections occurred shortly after an abortion or birth of a nonviable calf. Licking a fetus or calf or ingestion of feed contaminated by fetal fluids or vaginal exudates were the most probable sources of exposure. The external surface of only a few fetuses and calves was cultured before they were cleaned by the dam or grossly contaminated by debris. However, Brucella was recovered from the external surface of one aborted fetus and the fetal membrane and fluid of another. In addition to the oral route being of major importance in the bovine,3 7 other portals of entry are the conjunctiva7 23 and the unbroken or abraded skin.23 Some elk infections may have occurred through invasion of these structures following splashing of fluids or exudates. Elk are fastidious animals and invariably consumed the placenta. On one occasion a cow which had just aborted was observed to eat the placenta without allowing it to fall to the ground. Cow elk also attempted to eat aborted fetuses, and many fetuses were severely mutilated prior to discovery. This behavior also has been observed among free-ranging elk on winter feedgrounds. On several occasions during the study, cow elk were determined to be pregnant but no fetus or calf belonging to that particular individual was ever identified. We suspect that the entire fetus was consumed without our being aware of the abortion. This behavior could inadvertently reduce environmental contamination with Brucella but might serve also to spread the disease among elk if cows other than the dam take part. On two occasions, while abortions were occurring, other cows were observed to closely investigate and possibly lick the fetus before it had been fully expelled from the vagina. Chorioallantoic fluid of one of six fetuses, from (average 186) days gestation from Brucella positive elk, was culture-positive at necropsy. The cow with positive chorioallantoic fluid at approximately 200 days of gestation aborted her first calf, raised a second calf and was pregnant with a third calf when examined at necropsy. This fluid probably is the most important source of environmental contamination by elk. Lambert et al. 5 stressed the importance of contaminated mucus discharged from the genital tract of infected domestic cows as a source of infection. Vaginal exudates from four elk which had aborted or had nonviable calves were culture-positive for Brucella up to 17 days following parturition. Vaginal cultures were positive for Brucella from one cow at 6 and 9 days following two normal parturitions. Venereal transmission of brucellosis did not seem to be important among elk.

8 286 Journal of Wildlife Diseases Vol. 14, July, 1978 Uninfected cow elk were successfully bred six times over a 3-year period by infected bulls without contracting the disease. Another cow confined with infected bulls became sero-positive 2 months following the breeding season and may have become infected venereally, but no fetus was present at necropsy. A second cow in this group developed a relatively low transient titer in July, but the infection was not confirmed bacteriologically. The source of transmission of that infection could not be determined. If the infection was acquired venereally, it would have been during the previous breeding season (September-October) with an incubation period of approximately 10 months. Schroeder #{176} felt that leakage from the penis of infected domestic bulls resulting in contamination of food or water was the most important means of spread to susceptible cows from infected bulls. Licking infected semen as it leaks from cows bred by infected bulls may be a means of spread,23 and such an occurrence might have been responsible for either of these unexplained female elk infections. Brucella was recovered at necropsy from the genital tract of 9 of 17 infected bulls 1-year old or older. Brucella also was recovered from a seminal vesicle of a 4-month old calf which had numerous culture-positive tissues at necropsy. The organism was isolated from one of six semen samples obtained from two infected bulls during the breeding season. Although Brucella frequently has been isolated from semen of domestic cattle, infected bulls have not been important in the spread of the disease through natural service.29 Although the udder is a frequent site of localization of Brucella in cattle, 5 this did not seem to be the case in elk. Brucella was obtained from the udder of only 22% of 41 culture-positive cow elk 1-year or older at necropsy. Brucella was recovered from the milk of a single cow which was nursing a sero-negative calf. The organism was not isolated from six milk samples obtained from four other infected lactating elk. Brucella was not isolated from 92 cultured fecal samples from sero-positive elk calves up to 12 weeks of age which were known or assumed to have been born to infected cows. The organism was recovered from the feces of three nonviable calves at necropsy. Since none of these calves had nursed, the Brucella in their feces was felt to be due to exposure in utero. Environmental contamination via calf elk feces was not considered important. In some uninoculated elk, infections were contracted when a source of exposure could not be determined. Undetected abortions may have been the source of infection in a cow and a bull which became sero-positive in April of two separate years. Other infections were first detected in January and February. These may have been due to exposure the preceeding spring resulting in prolonged incubation periods. Clinical Effects of Brucellosis Abortion, premature birth or birth of nonviable calves were the most striking and important symptoms of brucellosis in elk. Of 29 cows which were pregnant when or after they became infected, 14 (48%) lost a calf from which Brucella was recovered. Seven (43%) of 16 artificially infected and 7 (54%) of 13 naturally infected cows lost their calves. One (11%) of nine cows which were followed through a second pregnancy lost a second calf and one (20%) of five lost a third calf. The same cow lost her second and third calf. Only calf losses proven to be due to brucellosis by recovery of Brucella and elimination of other causes of abortion were included in the number losing calves. Therefore, these rates of abortion are minimal. There was one mummified fetus and one fetus from which only the head was recovered. In neither case could the cause of death be determined. One calf died due to dystocia. As mentioned

9 Journal of Wildlife Diseases Vol. 14, July, previously, we suspected several aborted fetuses were never located, and if this was the case, the rate of abortion might have been higher. In 1972, seven cows were artificially inoculated at 5 months of pregnancy and seven cows at 6.5 months of pregnancy. Six of those inoculated at 5 months became infected prior to parturition and four lost calves, while no calves were lost due to brucellosis by those inoculated at 6.5 months gestation. Although the number of elk which were similarly inoculated at two stages of pregnancy i$ small, the data suggest that earlier exposure is more likely to result in premature termination of pregnancy. Cunningham5 stated that exposure of nonimmunized pregnant domestic cattle almost certainly leads to abortion. Stableforth and Galloway3 reported an outbreak of brucellosis in a previously uninfected herd would result in 40-50% abortion with the loss possibly reaching 100%. A challenge dose of 146 X 10 B. abortus strain 544 administered in the conjuctival sac at mid-pregnancy resulted in 90% abortion.2 In other studies the rates of abortion among pregnant cattle following initial infection were approximately 7Q%,8 73%27 and 85%.l8 The detected rate of abortion among pregnant elk in this study was somewhat lower than might have been expected with domestic cattle. The degree of exposure in cattle is important in determining whether infection will take place, determining the incubation period and determining the rate of abortion.2 In this study the number of organisms to which naturallyinfected elk were exposed was variable and unknown; the higher rate of abortion among naturally-infected elk possibly was due to exposure to more than the 7.5 X 10 organisms used in artificial infections and to exposure at times more likely to cause abortion. The infective dose used in this study resulted in 100% infection, rather than a desired iate of somewhat less than 100%, but an abortion rate of only 43% in artificially-infected elk. The infective dose was tenfold greater than required to establish an average infection in cattle, and it appears that elk may have a higher resistance than cattle to B. abortus type 1. Retained placenta was not observed in elk in this study, although retained placentas and subsequent secondary uterus infections frequently are reported in rangiferine2t and bovine brucellosis, and Cunningham5 wrote that retention in the domestic cow is related to prematurity of parturition. A single elk cow aborted once and failed to become pregnant through three succeeding breeding seasons. At necropsy, a large, heavily encapsulated abscess and adhesions involving the uterus, urinary bladder and parietal peritoneum of the ventral abdominal wall were found. Brucella and Staphylococcus epidermidis were recovered from the abscess. This cow s 3 years of infertility may have resulted from an injury or infection at the time of abortion. Brucellosis in the male bovine often causes orchitis, and swelling of the scrotum may be present due to distension of the cavity of the tunica vaginalis with exudate. 2 Rankin29 studied 12 naturally infected bulls in which enlargement of the seminal vesicles was the most frequent abnormality, with evidence of infertility in only one. Orchitis also has been reported in bison infected with B. abortus4 3 and caribou infected with B. suis.25 Seventeen bull elk over the age of 1 year were artificially- or naturallyinfected in this study. Brucella was not recovered from six at necropsy although the organism previously had been demonstrated by hemaculture from one. None of the 17 brucellosis-infected bulls had clinical signs of brucellosis. At necropsy Brucella was isolated from one or both epididymides of four, seminal vesicles of seven and ampullae of six. Jubb and Kennedy 2 stated that admixing of inflammatory products with semen may result in sterility. There was

10 288 Journal of Wildlife Diseases Vol. 14, July, 1978 no clinical indication of infertility among the 17 bull elk in this study, with the possible exception of the first breeding season among the three artificially infected bulls used to determine if venereal transmission might occur. None of the five cows confined with these bulls calved that year. The bulls were inoculated a month before the initiation of breeding season, and acute genital infections and pain may have prevented attempts to breed. Excitement or stress associated with being recently moved to a new corral and exposed to more activity also could have interferred with normal breeding activities. Carpal bursitis or hygroma is fairly common in cattle 2 and seemed to occur frequently in confined elk at Sybille. Secondary infections of hygromata by Brucella occurs in both cattle 2 and elk. Brucella infection of hygromata was the second most frequent sign seen in elk. Although generally associated with chronic stages of the disease, Brucella infected hygromata were present in three mature elk with serotiters of 1, 4 and 6 months duration. Brucella was recovered at necropsy from hygromata in 12 of 65 (18%) infected elk of 6 months of age or older. Several elk developed large and readily observable hygromata, but many were small and not detected until necropsy. Brucella infected hygromata seemed to cause little or no pain to the elk and the swelling (caused by accumulation of fluid and debris in the carpal bursa) often would recede spontaneously. Brucella was grown from fluid aspirated from a hygroma of a yearling cow but was not recovered from the much diminished hygroma at necropsy 2 years later. Inflammation of synovial membranes with distension of joint cavities and tendon sheaths also was a common sign in elk. Generally this was an indication of chronic brucellosis but was present in one 10-month old calf and in a yearling cow with a known brucellosis infection of less than 1-month duration. The fetlock joint and sheath of a deep flexor tendon of either fore or hind legs were most commonly affected. Swelling often was visible and frequently would recede spontaneously. Lameness often was severe and sometimes more than one limb would be affected simultaneously. The most severely affected elk underwent extensive weight loss. Of 65 infected elk examined at necropsy at an age of 6 months or more, Brucella was recovered from one or more joints or tecdon sheaths of 13 (20%). Joints and tendon sheaths from which Brucella was isolated were the anterior fetlock, posterior fetlock, carpal joint, stifle joint, anterior flexor tendon sheath, posterior flexor tendon sheath (two isolates each) and hock joint (one isolate). The pathologic changes observed in elk will be described in detail in a subsequent publication. Brucellosis in Elk Calves The bovine calf may be born infected due to Brucella bacteremia in utero, aspiration of amniotic fluids, or become exposed immediately after birth through ingestion of the organisms in colostrum and milk However, bovine calves generally are resistant and the infection is usually rapidly lost, at least partially through the activity of colostral antibodies, and they remain resistant until puberty Some calves born to infected cows may in fact maintain a dormant infection until some time later in life. lb,29 Elk calves born to infected cows in this study were quite variable in their response to brucellosis. Fifty-five calves born during the study survived, and blood samples were taken from most of them during the first or second day of life. Thirty-seven were born to Brucella infected dams, and 29 (89%) of them had a positive serotiter when the first blood sample was obtained. In most of these calves, the postnatal titer gradually declined. One female calf had a serotiter at 27 days of age and maintained an active

11 Journal of Wildlife Diseases Vol. 14, July, infection until killed at 4 years of age. The dam had been artificially inoculated with Brucella 2 months prior to parturition but demonstrated no infection until 27 days postpartum. Twenty-nine calves were held in the study beyond 6 months of age and an active infection had not been demonstrated by that age. Eleven of these were calves which had lost a postnatal titer. Six (four females and two males) of these 31 calves became infected between 6 and 12 months of age. One male and nine females remained uninfected when killed or removed from the study at 8-12 months of age. One male and four females of the remaining elk born in the study became infected (the infection was not confirmed bacteriologically in one of each sex), and six males remained uninfected when removed from the study at an age of 12 to 24 months. An active infection was not demonstrated until 3 years of age in one male and one female. Duration of Infection Fifty-six months was the longest interval from initial infection to recovery of Brucella at necropsy. This was in one of the original cows artificially-infected in February, 1972 which maintained a significant titer until necropsy. She aborted her first calf following infection and successfully raised four subsequent calves. In most cases it was not possible to determine the maximum duration of the disease because the elk were killed while infected. Bacteremias were intermittent and most frequent early in the course of the disease and were detected from 1 to 68 weeks (average for 23 elk -19 weeks) P1. One female born during the study aborted each fetus for 3 years and was extremely lame when examined at necropsy at 48 months of age. Hers was the most severe infection observed, and probably would have terminated in death due to malnutrition secondary to debilitation caused by Brucella induced arthritis and tendovaginitis. Five elk (two male and three females) apparently were able to overcome the infection. These elk had significant serotiters with durations of 31 to 65 weeks. Serotiters had been low or intermittently negative for periods of 9 to 37 months at the time they were killed. All were bacteriologically negative at necropsy. Another three elk also developed low or negative titers except that they maintained a significant CF T titer. Brucella was recovered from these elk at necropsy. Cattle occasionally become sero-negative after having been serologically reactive.2 Acknowledgements Sincere appreciation is expressed to Mr. George M. Thomas, Microbiologist, Veterinary Services, Animal and Plant Health Inspection Service, USDA, Laramie who performed the serologic tests and assisted with the bacteriology; to Mr. Thomas E. Heide and Mr. Carl V. Engstrom of the Sybille Game Research Unit who assisted with elk handling and sampling procedures, elk necropsy and daily care of the elk; to Dr. W. C. Ray, Brucellosis Epidemiology, Veterinary Services, Animal and Plant Health Inspection Service, USDA; and Mr. William G. Hepworth, Wyoming Game and Fish Department Research Laboratory for technical advice and assistance in carrying out the study and for review of the manuscript. Cultures of B. abortus type 1 strain 2308 were supplied by Biologic Reagents Section, Veterinary Services, Diagnostic Laboratory, Ames, Iowa, This study is a contribution of Wyoming Game and Fish Department Federal Aid to Wildlife Restoration, Projects FW-3-R and W-53-D, and was partially financed by Veterinary Services, Animal and Plant Health Inspection Service, USDA.

12 290 Journal of Wildlife Diseases Vol. 14. July, 1978 LITERATURE CITED 1. BAKER, M.G., G.J. DILLS and F.A. HAYES Further experimental studies on brucellosis in white-tailed deer. J. Wildl. Manage. 26: BERMAN, D.T., M.R. IRWIN and B.A. BEACH Statistical considerations of controlled experiments in brucellosis. Am. J. vet. Res. 10: BIRCH, R.R. and H.L. GILMAN The channel of invasion of Bacterium abortum with special reference to ingestion. Cornell Vet. 15: CORNER, A.H. and R. CONNELL Brucellosis in bison, elk, and moose in Elk Island National Park, Alberta, Canada. Can. J. Comp. Med. and Vet. Sci. 22: CUNNINGHAM, B A difficult disease called brucellosis. In: Bovine Brucellosis, an International Symposium, Ed. by R.P. Crawford and R.J. Hidalgo, Texas A&M University Press, College Station. pp FENSTERMACHER, R and O.W. OLSEN Further studies of disease affecting moose III. Cornell Vet. 32: FITCH, C.P., LM. BISHOP, W.L. BOYD and M.D. KELLY Results of blood cultures on seven heifers artificially infected with Brucella abortus. J. Am. vet. med. Ass. 95: HUDDLESON, I.F Brucellosis in Man and Animals. Commonwealth fund. New York. 379 pp. 9. HUNTLEY, B.E., R.N. PHILIP and J.E. MAYNARD Survey ofbrucellosis in Alaska. J. Infect. Die. 112: JEWSON, W.L, C.W. FISHEL and E.L. CHEATUM Brucellosis in a moose Akes americanus. J. Wildl. Manage. 17: JONES, LM.,J.B. HENDRICKS and D.J. BERMAN The standardization and use of the complement-fixation test for the diagnosis of bovine brucellosis, with a review of the literature. Am. J. vet. Res. 24: JUBB, K.V.F. and P.C. KENNEDY Pathology of E)omestic Animals. Vol. 1. Academic Press, New York. 593 pp. 13. KATZ, J.S Brucellosis in wildlife. J. Am. vet. med. Ass. 99: LAMBERT, G., T.E. AMERAULT, C.A. MANTHEI and E.R. GOODE Immunogenic response of calves vaccinated at different ages with Brucella abortus strain 19. Proc. U.S. Livestock Sanitary Ass. pp Further studies on the persistence of Brucella abortus infection in cattle. Proc. U.S. Livestock Sanitary Ass. pp LAPRAIK, R.D., D.D. BROWN, H. MANN and T. BRAND Brucellosis: A study of five calves from reactor dams. Vet. Rec. 97: MANTHEI, C.A Summary of controlled research with strain 19. Proc. U.S. Livestock Sanitary Ass. pp: and RW. CARTER Persistence of Brucella abortus infection in cattle. Am. J. vet. Res. 11: MARTIN, W.H. and W.H. RITCHIE The microtiter technique for the diagnosis of bovine anaplasmosis. Proc. 6th Nat l Anaplasmosis Conf., Las Vegas, Nevada. pp MCCULLOUGH, N.B., C.W. EISELE and A.F. BYRNE Incidence and distribution of Brucella abortus in slaughtered Bang s reactor cattle. Public Health Rpt. 66:

13 Journal of Wildlife Diseases Vol. 14, July, MCEWEN, A.D., F.W. PRIESTLEY and J.D. PATERSON An estimate of a suitable infective dose of Br. abortus for immunization tests on cattle. J. Comp. Path. and Therapeutics. 52: MURIE, O.J The Elk of North America. The Stackpole Co., Harrisburg, Pa. and Wildl. Manage. Inst., Washington, D.C. 376 pp. 23. NATIONAL RESEARCH COUNCIL Brucellosis research: An evaluation. National Academy of Sciences, Washington, D.C. 186 pp. 24. NEILAND, K.A Rangiferine brucellosis in Alaskan canids. J. Wildl. Dis. 6: Further observations on rangiferine brucellosis in Alaskan carnivores. J. Wild!. Dis. 11: , J.A. KING, B.E. HUNTLEY and R.O. SKOOG The diseases and parasites of Alaskan wildlife populations, Part I. Some observations on brucellosis in caribou. Bull. Wild!. Dis. Ass. 4: PLENDERLEITH, R.W.J Some observations on brucellosis in a Jersey herd, Vet. Rec. 87: PLOMMET, M Studies on experimental brucellosis in cows in France. In: Bovine Brucellosis, an International Symposium. Ed. by R.P. Crawford and R.J. Hidalgo. Texas A&M University Press, College Station. pp RANKIN, J.E.F Brucella abortus in bulls: A study of twelve naturally infected cases. Vet. Rec. 77: SCHROEDER, E.C Bureau of animal industry investigations on bovine infectious abortion. J. Am. vet. med. Ass. 60: STABLEFORTH, A.W. and l.a. GALLOWAY Diseases Due to Bacteria. Vol. 1. Academic Press, New York. pp TABER, R.D Criteria of sex and age. In: Wildlife Management Techniques. Ed. by R.H. Giles. The Wildl. Soc., Washington D.C. 391 pp. 33. THORNE, E.T., J.K. MORTON and G.M. THOMAS Brucellosis in elk. 1. Serologic and bacteriologic survey in Wyoming. J. Wildl. Dis. 14: TUNNICLIFF, E.A. and H. MARSH Bang s disease in bison and elk in Yellowstone National Park and the National Bison Range. J. Am. vet. med. Ass. 86: U.S. Department of Agriculture (not dated). Standard agglutination test procedures for the diagnosis of brucellosis. National Animal Disease Laboratory Diagnostic Reagents Manual 65D. ARS, ANH, Natn l. Anim. Die. Lab., Ames, Iowa (not dated). Supplemental test procedures for the diagnosis of brucellosis. National Animal Disease Laboratory Diagnostic Reagents Manual 65E. ARS, ANH, Natn l. Anim. Dis. Lab., Ames, Iowa. 37. WITTER, J.F. and D.C. O MEARA Brucellosis. In: Infectious Diseases of Wild Mammals. Ed. by J.W. Davis, L.H. Karstad and D.O. Trainer. Iowa State University Press, Ames. pp YOUATT, W.G. and LD. FAY Experimental brucellosis in whitetail deer. Am. J. vet. Res Received for publication 5 December 1977