Elk Brucellosis Surveillance and Reproductive History

2013-14 Elk Brucellosis Surveillance and Reproductive History Neil Anderson, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Kelly Proffitt, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Julee Shamhart, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Torrey Ritter, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Tom Chandler, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Jennifer Ramsey, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Keri Carson, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Jennifer Jones, Montana Fish, Wildlife and Parks, 1400 South 19 th Ave., Bozeman, MT 59718. Montana Fish, Wildlife and Parks (MFWP) has conducted surveillance for brucellosis in elk populations since the early 1980s. Surveillance consisted of screening blood serum for antibodies against exposure to Brucella. Elk that test positive for exposure to Brucella (seropositive) may or may not be actively infected with the bacteria. Although not a true indicator of infection or the ability of an animal to shed Brucella on the landscape, detection of seropositive elk indicates Brucella is present in the area and suggests that the disease could be circulating within the elk population, particularly if there are no other known sources for the disease. Prior to 2000, brucellosis had been documented in 5 hunting districts (HDs) in the Greater Yellowstone Area (GYA). Based on elk movement patterns, the distribution of brucellosis in elk was believed to be limited to all or portions of 11 HDs in the GYA (Figure 1). Although testing focused on the GYA, additional testing was conducted in much of the state with no brucellosis being detected (Figure 2). In 2008, MFWP expanded surveillance efforts to include 30 HDs within the GYA, partially in response to cattle cases in 2007 and 2008. Efforts focused on the collection and testing of blood from hunterharvested elk and the opportunistic testing of animals captured and sampled as part of research projects. MFWP continued this effort until the winter of 2010/11 and evaluated the effectiveness of the surveillance program. Across 3 years (2008-2010), sample sizes within the 30 individual HDs varied widely from 2 to 229. Although data was insufficient in some HDs to evaluate brucellosis presence/absence, additional insight on the distribution and prevalence of the disease was obtained. Prevalences appeared to be increasing in areas where adequate information was available and comparisons could be made to historical data. The disease was also detected in areas outside of its previously documented distribution (Anderson et. al. 2009, Anderson et. al. 2010). However, the small number of samples obtained in many hunting districts did not achieve the goal of delineating the geographical boundary of brucellosis in Montana elk populations. 1

As a result, MFWP initiated the current multi-year targeted surveillance and research project in the winter of 2010/2011. The goals of the project were to increase our understanding of the geographical distribution of brucellosis, how the disease functions in elk populations, the transmission risk seropositive elk pose to livestock and other elk populations, and the potential movement pathways for brucellosis between elk populations. In order to achieve these goals, MFWP proposed to capture and screen approximately 100 adult female elk in areas adjacent to the known distribution of brucellosis, with relatively large elk populations and significant livestock concerns. Up to 30 elk per herd received a global positioning systems (GPS) collar. Study areas were identified through collaboration between MFWP and the Department of Livestock staff. Figure 1. The distribution and seroprevalence of brucellosis in elk populations from 1981-2000. 2

Figure 2. Brucellosis surveillance results for elk tested from 1981-2014. Results were based on screening of blood samples from hunter harvested and research elk for exposure to Brucella. Only hunting districts with > 10 samples from adult female elk are depicted. Study areas and methods for targeted surveillance and research project: Elk were captured and tested in 6 different areas in western Montana from January 2011 through February 2014. These areas consisted of the Blacktail/Sweetwater Creek (Blacktail) area in hunting districts 324 and 326, Sage Creek/Clark Canyon (Sage Creek) areas of hunting district (HD) 325, south Bannack area in HD 329, southern and western Pioneer Mountains (Pioneers) in HDs 329, 331 and 332, the southern and western Tobacco Roots in HDs 320 and 333, and the Red Mountain and Black s Ford areas of HD 311 (Figure 3). Blood was collected and serum samples initially screened in the field utilizing the Card and/or the Fluorescent Polarized Assay (FPA) tests. Samples were then retested at the Montana Department of Livestock Diagnostic Lab (Diagnostic Lab). Final classification of sero-status was based on test results received from the Diagnostic Lab. GPS radio collars were placed on approximately 30 elk (both seropositive and seronegative elk) from each herd in order to track movements and evaluate risk of brucellosis transmission to livestock and other elk populations (Proffitt et al. 2012, Proffitt et al. 2013). Seropositive elk were recaptured and 3

retested every year. If pregnant, seropositive elk also received a vaginal implant transmitter (VIT) in order to detect birth events and collect birth/abortion tissues for. VITs are programmed to emit a slow pulse when the temperature is 32⁰ C or higher, emitting a faster pulse once the temperature cools below 28⁰ C. This allows field crews to determine if an implant has been expelled. However, if the VIT is expelled and the ambient temperature is above 32⁰ C, or if the implant is in the sun elevating the VIT temperature above 32⁰ C, the VIT will reset and emit the slow pulse. The implants also have a precise event transmitter (PET) code which indicates the time since the VIT was expelled and cooled to a temperature below 28⁰ C. However, the code will reset and start a new cycle if the temperature rises above 32⁰ C again, resulting in an inaccurate time of expulsion. Due to that possibility, both the elapsed time from when the elk was last located with a retained VIT and when the VIT was recovered, and the PET code time were reported. After 5 years, all remaining radio-collared, seropositive elk will be euthanized and tissues d to determine if they are infected with Brucella. The project is currently in its fourth year. Results for the ongoing surveillance and reproduction study of seropositive elk are presented in Appendix A and B, respectively. Figure 3. The location of the targeted elk surveillance project study areas during 2011-2014. 4

Appendix A: Elk Brucellosis Surveillance Results and Discussion The goal of the surveillance portion of the targeted surveillance and research project is to delineate the geographic distribution of the disease in elk populations. This has been accomplished by focusing disease screening efforts on different areas just outside the known distribution of brucellosis. A secondary goal is to obtain baseline seroprevalence data when the disease is detected. When exposure to Brucella was detected during targeted surveillance, prevalences varied from 5.4% in the Sage Creek study area to 22.5% in the Black s Ford area (Table 1). Seroprevalence was also higher than observed during the early 1990 s in the Gardiner and Madison Valley areas, where the disease has historically been detected. Seroprevalences in the late 1980 s and early 1990 s in these areas averaged less than 2% (Anderson et al. 2010). Within the GYA, seroprevalences ranged from 4.0% - 30.7% when all forms of sample collection (hunter harvest, targeted surveillance and research) from 2008 through 2014 were pooled (Figure 4). Seroprevalence in HD 323 was the highest observed, but based on only 13 samples obtained from hunter-harvested elk from 2008 2010. This is not considered a large enough sample for an accurate prevalence estimate with upper and lower 95% confidence intervals of 12.7% and 57.6%, respectively. Seropositve elk were not detected in the south Bannack, Pioneers or Tobacco Root study areas. Additional surveillance was conducted in the Bitterroot Mountains (n = 123, 2010-2013), Missouri River Breaks (n = 53, 2013), and the Sapphire Mountains (n = 45, 2014) in conjunction with research projects. No seropositive elk were detected in these areas. Table 1. Targeted surveillance for brucellosis in MT elk populations, Jan 2011 Feb 2014. Results are based on elk captured for targeted surveillance and do not include additional samples collected from hunter-harvested animals. Study Area Hunting Districts Sample Size Seropositive (%) 95% Confidence Interval Blacktail 324, 326 100 12 (12.0%) 7.0% - 19.8% Sage Creek 325 93 5 (5.4%) 2.3% - 12.0% South Bannack 329 30 0 0% -11.4% Pioneer Mtns 329, 331, 332 100 0 0% - 3.7% Tobacco Root Mtns 320, 333 70 0 0% - 5.2% Red Mtn 311 20 1 (5.0%) 0.2% - 23.6 Black s Ford 311 40 9 (22.5%) 12.3% - 37.5% 5

Figure 4. The general geographical distribution and seroprevalence for brucellosis in Montana elk populations as determined from samples collected in 2008-2014. Samples from elk captured for targeted surveillance and research and hunter-harvested elk were pooled when available. Capture and testing of elk has occurred in 6 different areas over the four years of the project, as specified in the project proposal. Detection of seropositive elk in the Blacktail, Sage Creek, Red Mountain and Black s Ford areas expanded the known distribution of brucellosis in elk populations. The documented expansion of the elk brucellosis distribution may be attributed to a combination of factors including increased surveillance efforts, changing elk distributions, and movement of the disease into new population segments. The lack of detection of seropositive elk in the Pioneer and Tobacco Root Mountains increases our confidence that elk in these areas present little risk of transmitting the disease to livestock. 6

Appendix B: Seropositive Reproduction Study A primary goal of the targeted elk brucellosis surveillance and research project for brucellosis in elk is to evaluate the risk seropositive elk pose for shedding Brucella abortus on the landscape. Currently there is no literature indicating long term effects of B. abortus on elk pregnancy. It has been assumed that brucellosis in elk would behave similar to what has been observed in cattle and bison. In order to determine the fate of pregnancies and the ability of seropositive elk to shed B. abortus on the landscape through birth events, seropositive pregnant elk are fitted with a vaginal implant transmitter (VIT) and monitored closely over the course of a pregnancy. When a birth event occurs, the area is searched for an aborted fetus and birth material. The fetus (if an abortion occurred), birth material, environmental samples identified as having potential contact with birth material and fluids, and the VIT are collected and submitted for. Seropositive elk will be recaptured and, if pregnant, fitted with a VIT for five years, after which they will be removed from the population. Tissues from seropositive elk will be collected and d at this time to determine if they are infected with B. abortus. Blood serum from elk captured during the study was screened in the field utilizing the Card and/or the Fluorescent Polarization Assay (FPA) for potential exposure to Brucella. Elk in the Blacktail (n=8), Sage Creek (n=5) and Black s Ford (n= 6) study areas were initially identified as being seropositive based on field tests in January 2011, January 2012 and February 2014, respectively. These elk received a GPS collar, which also emits a VHF signal, so they could be relocated. If pregnant they received a VIT and were monitored approximately 2 times per week until either the VIT was expelled or the end of June. After the end of June, if an elk had not expelled the VIT, relocation frequency was reduced to approximately once every 2 weeks. The majority of elk gave birth prior to the end of June. Twelve elk were identified as being seropositive on field tests in the Blacktail and Sage Creek study areas and 4 additional seropositives in the Blacktail study area were identified later when serum was retested at the Diagnostic Laboratory. These 4 animals were not identified or collared in the field and, therefore, not included in the reproduction study. One collar failed during the first season and the animal could not be relocated. Two elk died during the course of the study. The remaining 10 elk initially identified as seropositive during field testing in the Blacktail and Sage Creek study areas have been recaptured, retested and fitted with an implant (if pregnant) for 4 and 3 years, respectively. In 2014, 6 additional elk were identified as being seropositive during field testing in the Black s Ford area. Four of these were pregnant and fitted with an implant. Three additional elk were identified as being positive for exposure to brucellosis when serum samples were screened at the Diagnostic Laboratory. Two of these seropositive elk were radiocollared, by chance, and will be recaptured in 2015 and included in the seropositive elk reproduction study. In 2014, 12 seropositive pregnant elk were located and the status of the VIT verified, utilizing telemetry equipment, an average of twice/week from February 19 th until parturition or the end of June, whichever came first. After June 30, elk that still retained a VIT were tracked about once every two weeks. The elapsed time (the time between the last location when the elk was carrying the VIT and when the expelled VIT was recovered) ranged from 1 to 17 days. Ten of 12 VITs were recovered within 5 days of the implant being expelled (Table 2). Two VITs were recovered after June 30 th, with an elapsed 7

time of 15-16 days. BT10045 s implant was recovered on July 16 th with a recently born calf (estimated to be within two days) near the birth site. SC11045 s implant was recovered September 15 th. She was last located July 30 th and was carrying the implant at that time. The elapsed time does not necessarily indicate how long the implant was on the landscape since being expelled. The birth event occurred at some point during that time period. PET codes provide a better indication of how much time has passed since the implant was expelled (see methods for a more detailed explanation). PET code times ranged from 0.5 20.5 hrs, when available, suggesting most birth sites were visited within 24 hours of the VIT being expelled (Table 2). Table 2. Vaginal implant transmitter (VIT) recovery times from seropositive pregnant elk in Montana, 2014. The elapsed time is the period of time (days) between the last location when the VIT was still being retained by the cow elk and when the VIT was recovered after being expelled. The PET code is the estimated time (hrs) for VIT recovery after being expelled and cooling below 28⁰ C. Times are based on the precise event transmitter (PET) code programmed into the implant. Elk Elapsed time PET code time (hrs) Elk Elapsed time PET code time (hrs) BF13004 3 0.5 BT10068 2 13.5 BF13027 5 11.5 BT10083 1 1.0 BF13039 1 19.5 SC11031 1 6.0 BF13073 2 NA Temp above 32⁰ C SC11045 15 NA Temp above 32⁰ C BT10045 17 NA Temp SC11050 3 4.0 above 32⁰ C BT10058 2 20.5 SC11087 5 9.0 The pregnancy status of 18 individual seropositive elk was evaluated a total of 46 times during 2011-2014. Thirteen elk were not pregnant during this time period resulting in a total of 33 pregnancies being monitored during the first 4 years of the study. Excluding VIT failures (n=1), mortalities (n=2) and the retention of a VIT (n = 1), 29 reproductive years were evaluated to determine the ability of seropositive elk to shed B. abortus on the landscape. At each birth site, the VIT was located and the area searched for a fetus and birth material. The VIT, any birth material, and environmental samples potentially containing birth fluids were collected and submitted for. Tissues collected at birth sites varied from intact fetuses to VITs only. The majority of birth sites visited after May 15 consisted of a bed site, the VIT (in close proximity to the bed site), and wet or damp areas which may have been from amniotic fluid. Only occasionally were we able to find birth tissue and it was generally small in size and quantity. VITs were collected within 5 days of being expelled in 24 (83%) of the 29 reproductive years during the course of the study. Sixty-two percent (18/29) of the VITs were recovered within 2 days of being expelled. Tissues collected during 2011-2014 from a total of 29 birth sites were d. All birth sites sampled after May 18 th (n =26) were negative for B. abortus (Table 3). B. abortus was recovered from samples collected at 3 birth sites during the study. The aborted fetus of SC11087 was collected on 8

April 25 th, 2012 and BT10045 s fetus was collected on May 17 th, 2012. Both were positive for B. abortus (Anderson et al, 2012). BF13027 expelled her VIT on March 30 th, 2014 but a fetus could not be located. B. abortus was d from the implant suggesting an abortion event occurred. In all three cases in which B. abortus was d, the birth event occurred prior to May 18 th. We defined a fullterm pregnancy as a birth event occurring after May 15 th. Detection of B. abortus in a fetus after May 15 indicates there is some risk of an abortion occurring during a full-term pregnancy, as defined for this project. In all cases in which a live calf was delivered, or the status of the calf was not determined (no abortion was detected), B. abortus was not d from samples collected. Initial indications are that the majority of pregnant seropositive elk were able to carry calves to full term. However, the ultimate fate of most calves was not known and it is unknown if survival rates of calves of seropositive mothers are similar to survival rates of calves from seronegative mothers. More research is needed to evaluate if there are differential survival rates. If each birth event is considered an independent observation, the odds of B. abortus being shed during an abortion from seropositive elk was approximately 1 out of every 9 birth events. However, abortions were only detected in 2 of the 4 years. Our ability to determine if an abortion had occurred was hampered by an implant failure for one reproductive year. In 2013, BT10045 s implant failed in early April. She was monitored frequently and was never observed with a calf, suggesting a reproductive failure. She aborted a positive fetus the previous year and it is possible that she aborted again in 2013, the VIT failing when expelled. A birth site could not be located or tissues collected due to the VIT malfunction. Three reproductive years were excluded from birth site analysis. BT10063 and SC11097 were both pregnant when they died in 2012 and 2013, respectively. Tissues from the reproductive tracts of these elk were collected from the carcass and d. B. abortus was not recovered. However, because they did not give birth, the data was excluded from our birth site summary. SC11050 was pregnant based on rectal palpation and a blood test in 2013. However, she did not expel her implant. The implant was recovered in 2014 when she gave birth to a live calf. The 2013 pregnancy was also excluded from analysis. Three elk have had long term reproductive complications, potentially related to exposure to B. abortus, but the actual cause is not known. BT10045 was not pregnant in 2011, aborted in 2012, and then had a possible abortion and VIT failure the next year in early April. In 2014 she retained her implant until mid July. A calf was present at the birth site, but was small in size and made no attempt to stand or use its back legs. It was weak in appearance and unlikely to survive. Samples collected at the 2014 birth site were negative on. BT10045 has not produced a viable calf since entering into the study in 2011. Two elk, BT10055 and BT10075, have not been pregnant in the 4 years of study. Reproductive tracts from these individuals will be examined when they are removed from the study to determine if there is a physiological reason why they have not been pregnant. 9

Table 3. Pregnancy status and results from birth sites of seropositive elk, 2011-2014. Elk with pregnancy status of open were not pregnant based on rectal palpation or ultrasound at time of capture. Elk ID # Original Capture Location 2011 Pregnancy Status 2011 Birth Site Results 2012 Pregnancy Status 2012 Birth Site Results 2013 Pregnancy Status 2013 Birth Site Result 2014 Pregnancy Status BT10055 HD 324/326 Open NA Open NA Open N/A Open NA 2014 Birth Site Result BT10045 HD 324/326 Open NA Abortion - B. abortus d VIT failure - possible abortion BT10068 HD 324/326 BT10075 HD 324/326 Open NA Open NA Open NA Open NA BT10058 HD 324/326 *BT10063 HD 324/326 / Mortality * NA NA NA NA BT10083 HD 324/326 *SC11097 HD 325 NA NA SC11050 HD 325 NA NA / Mortality * NA NA Did not expel VIT SC11087 HD 325 NA NA Abortion - B. abortus d SC11031 HD 325 NA NA Open NA SC11045 HD 325 NA NA Open NA BF13001 HD 311 NA NA NA NA NA NA Unknown ** NA BF13039 HD 311 NA NA NA NA NA NA BF13002 HD 311 NA NA NA NA NA NA Open NA BF13009 HD 311 NA NA NA NA NA NA Unknown ** NA BF13004 HD 311 NA NA NA NA NA NA BF13027 HD 311 NA NA NA NA NA NA Possible abortion - B. abortus d BF13061 HD 311 NA NA NA NA NA NA Open NA BF13073 HD 311 NA NA NA NA NA NA * Culture results of tissues collected from a carcass **Initial field test results based on the Card test were negative. Subsequent testing at the Montana Dept. of Livestock Diagnostic Laboratory was positive for exposure to Brucella. These elk were collared and will be recaptured and included into the seropositive reproductive study in 2015. 10

If Brucellosis affects elk pregnancy in a similar manner to cattle and bison, considering each birth event as an independent observation may not be appropriate in evaluating risk. Risk of B. abortus transmission would be greatest in the first pregnancy after infection and significantly less in subsequent pregnancies. Based on preliminary information obtained in this study, B. abortus (if present) was not present at detectable levels in samples collected from birth sites where elk carried a fetus to full term and no evidence of an abortion was observed. This suggests that seropositive elk giving birth to live calves likely pose little to no risk of transmitting brucellosis to livestock or other wildlife. Final determination of transmission risk from seropositive elk should not be made until the project is completed and additional information is obtained. Literature Cited: Anderson, N. J., J.M. Ramsey and K.D. Hughes, 2009. 2008 elk brucellosis surveillance final report. Montana Fish, Wildlife and Parks, Bozeman, Montana, 20 pp. Anderson, N. J., J. M. Ramsey, K. Hughes, K. Lackey and S. Olind. 2010. 2009-10 brucellosis surveillance in elk. MT Fish, Wildlife and Parks, Bozeman, MT. Anderson N., J. Shamhart, T. Ritter, J. Ramsey, K. Proffitt, K. Carson, C. Fager and B. Brannon. 2012. 2011-2012 Elk Brucellosis Survey and Research Summary. MT Fish, Wildlife and Parks, Bozeman, MT. Proffitt K., J. Shamhart and N. Anderson. 2012. Elk Movements and Brucellosis Risk in the Blacktail and Sweetwater Hills Area, HD 324 and HD 326. MT Fish, Wildlife and Parks, Bozeman, MT. Proffitt K., J. Shamhart and N. Anderson. 2013. Elk Movement and Brucellosis Risk in the Sage Creek and Clark Canyon areas. MT Fish, Wildlife and Parks, Bozeman, MT. 11