Experimental Infection of Mexican Free-Tailed Bats (Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus is thought to have originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies in the southern United States, often in urban areas and, as such, could be exposed to the virus from infected humans. We experimentally challenged wild T. brasiliensis with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of 10 bats oronasally inoculated with SARS-CoV-2, 5 became infected and orally excreted moderate amounts of virus for up to 18 days postinoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while T. brasiliensis are susceptible to SARS-CoV-2 infection, infection of wild populations of T. brasiliensis would not likely cause mortality. However, the transmission of SARS-CoV-2 from T. brasiliensis to or from humans, or to other animal species, is a possibility requiring further investigation to better define. IMPORTANCE As the COVID-19 pandemic has continued for 3+ years, there has been increasing concern that the SARS-CoV-2 virus will enter wildlife populations and potentially create new reservoirs where the virus could adapt to a new host and create variants. This is particularly possible with species that reside in man-made structures, in proximity to infected human populations. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies, often in urban settings and, thus, can be exposed by infected humans and potentially transmit the virus to new hosts. We experimentally challenged T. brasiliensis with SARS-CoV-2 and revealed that they are susceptible to the virus and excrete moderate amounts for up to 18 days postinoculation. This is important information for wildlife biologists, wildlife rehabilitation workers, and the general public that may contact these animals.

Experimental infection of Mexican free-tailed bats ( Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats ( Tadarida brasiliensis : TABR) live in large colonies in the southern United States, often in urban areas, and as such, could be exposed to the virus from infected humans. We experimentally challenged wild TABR with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of nine bats oronasally inoculated with SARS-CoV-2, five became infected and orally excreted moderate amounts of virus for up to 18 days post inoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while TABR are susceptible to SARS-CoV-2 infection, infection of wild populations of TABR would not likely cause mortality. However, the transmission of SARS-CoV-2 from TABR to or from humans, or to other animal species, is a distinct possibility requiring further investigation to better define.

Short communication: Attempts to identify Clostridium botulinum toxin in milk from three experimentally intoxicated Holstein cows.

Three adult lactating Holstein cows were injected in the subcutaneous abdominal vein with 175 ng/kg of body weight of Clostridium botulinum type C toxin (451 cow median toxic doses) to determine if this botulinum toxin crosses the blood-milk barrier. Whole blood (in sodium heparin) and clotted blood serum samples were taken at 0 min, 10 min, and 3, 6, 9, and 12 h postinoculation. Milk samples were taken at 0 min and at 3, 6, 9 and 12 h postinoculation. All samples were tested for the presence of the toxin using the mouse bioassay and immunostick ELISA test. The immunostick ELISA identified the toxin in whole blood and the mouse bioassay identified the toxin in serum at all times examined in all 3 animals. Toxin was not identified by either detection method in milk samples collected from the 3 animals. From these results, it appears that Clostridium botulinum type C toxin does not cross from the blood to the milk in detectable concentrations.

Serologic response of Rio Grande wild turkeys to experimental infections of Mycoplasma gallisepticum.

The serologic response of Rio Grande wild turkeys (Meleagris gallopavo intermedia) to Mycoplasma gallisepticum (MG) was determined. Free-ranging turkeys were caught in southern Texas, shipped to the University of Wisconsin, Madison, and housed in isolation facilities. Fourteen birds were exposed to MG, by intratracheal and intranasal inoculation. Eight birds received sterile broth only. Two wk prior to the end of the experiment, MG exposed turkeys were stressed by challenge with a serologically unrelated mycoplasma. Serum from all exposed birds reacted positively for MG antibody by the rapid plate agglutination (RPA) procedure within 2 mo postexposure (PE) and all but one remained positive for 14 mo PE. Less than one half of the exposed birds developed positive MG antibody titers detectable by the hemagglutination inhibition (HI) test within 2 mo PE, and by 10 mo PE, none had positive titers. Antibody was detected by the HI test in two of 11 infected turkeys, 14 mo PE, and titers increased significantly within 2 wk. MG was isolated from tracheal swabs from two infected birds 2 mo PE, but attempts thereafter failed. However, at the termination of the experiment 15 mo later, MG was isolated from lung tissue of three of 11 exposed turkeys and from a blood clot found in the lower trachea of one bird.

Brain acetylcholinesterase activity in botulism-intoxicated mallards.

Brain acetylcholinesterase (AChE) activity in captive-reared mallards (Anas platyrhynchos) that died of botulism was compared with euthanized controls. AChE levels for both groups were within the range reported for normal mallards, and there was no significant difference in mean AChE activity between birds that ingested botulism toxin and died and those that did not.

Effects of lead shot ingestion on selected cells of the mallard immune system.

The immunologic effects of lead were measured in game-farm mallards (Anas platyrhynchos) that ingested lead shot while foraging naturally, mallards intubated with lead shot, and unexposed controls. Circulating white blood cells (WBC) declined significantly in male mallards exposed to lead by either natural ingestion or intubation, but not females. Spleen plaque-forming cell (SPFC) counts were significantly lower in mallards intubated with lead pellets compared to controls. Declines in WBC and SPFC means with increasing tissue lead concentrations provide further evidence that lead exposure reduced immunologic cell numbers. Hormonal activity and diet may have influenced the immunologic effects of lead exposure in this study.

Use of sentinel mallards for epizootiologic studies of avian botulism.

Captive-reared mallards (Anas platyrhynchos) were used as sentinels to study the epizootiology of avian botulism at the Sacramento National Wildlife Refuge, Willows, California (USA) from 1986 to 1989. Sentinel mallards were wing-clipped, and 40 to 50 birds were confined in 1.6-ha enclosures in 11 selected wetlands (pools). Enclosures were searched intensively three to four times weekly from July through October. Sick and dead wild and sentinel birds were collected, necropsied, and tested for type C botulism toxin. Botulism epizootics occurred in sentinel mallards in 1986, 1987, and 1989, but only a few isolated cases of botulism were detected in 1988. In most epizootics, botulism also was detected simultaneously in wild birds using the same pool outside the enclosure. Epizootics in sentinels were initiated and perpetuated in the absence of vertebrate carcasses. A sex-specific trend in the probability of intoxication was detected, with males contracting botulism at a higher rate than females. Daily mortality rates of sentinels during botulism epizootics ranged from 0.0006 to 0.0600, with a mean of 0.0190. These rates would result in the daily loss of 0.6 to 60 birds per thousand at risk. The use of sentinel birds provided an effective means of gathering site-specific epizootiologic data.

Hematozoan parasites of Rio Grande wild turkeys from southern Texas.

One hundred twenty-three of 300 blood samples (41%) taken from Rio Grande wild turkeys (Meleagris gallopavo intermedia) from three locations in southern Texas (Welder Wildlife Refuge, Chaparrosa Ranch, and Campo Alegre Ranch) and subinoculated into domestic broad-breasted white turkey poults were positive for a Plasmodium (Novyella) sp. Analysis of blood films from 350 turkeys revealed Haemoproteus meleagridis in 76% of the birds. A significantly greater mean parasite intensity was observed in birds from Welder Wildlife Refuge. Birds from the Campo Alegre Ranch exhibited a significantly higher prevalence of H. meleagridis than birds from Chaparrosa. The Plasmodium sp. was infective for canaries (Serinus canaria), bobwhites (Colinus virginianus), and ring-necked pheasants (Phasianus colchicus), but would not produce infection in white leghorn chickens (Gallus gallus) or Coturnix quail (Coturnix coturnix). Attempts to infect Culex tarsalis and C. pipiens were unsuccessful. Asexual erythrocytic synchrony was not observed when blood-induced infections were monitored in two domestic turkey poults every 4 hr for 72 hr. Exoerythrocytic stages were not found upon examination of impression smears and tissue samples taken from brain, liver, spleen, kidney, lung, and bone marrow. The Plasmodium sp. is most similar morphologically to three species in the subgenus Novyella, P. hexamerium, P. vaughani, and P. kempi. The most striking similarities are to P. hexamerium, and involve mean merozoite number, erythrocytic schizont location, and vertebrate host susceptibility. It differs from P. vaughani in being able to infect turkeys and in type of parasitized erythrocytes. Differences to P. kempi include mean merozoite number, and ability to infect pheasants, and its inability to develop in C. pipiens and C. tarsalis.

Experimental Mycoplasma gallisepticum infections in captive-reared wild turkeys.

The effects of Mycoplasma gallisepticum (MG) infections on egg production, fertility, and hatchability were studied in captive-reared wild turkeys (Meleagris gallopavo). Three groups of adult birds, each consisting of four hens and two toms, were exposed to MG by the respiratory route at the beginning of their breeding season. Fourteen control birds received sterile growth medium. Although no mortality of infected or control birds occurred, egg production during the first breeding season after infection was reduced. The mean number of eggs/hen/day produced by infected groups the first breeding season postexposure (PE) was significantly lower than the control value. The mean number of eggs produced daily by the same hens 1 yr later was unaffected by MG infection. The percentage of fertile eggs produced by infected groups was slightly reduced in both the first and second breeding seasons PE. Hatchability of fertile eggs from infected hens was significantly lower than eggs from control hens. Productivity may be impaired if MG infections occur in free-ranging wild turkey populations.

Detection of Clostridium botulinum type C cells in the gastrointestinal tracts of Mozambique Tilapia (Oreochromis mossambicus) by polymerase chain reaction.

We established a method of directly detecting Clostridium botulinum type C cells, while minimizing spore detection, in the intestinal contents of Mozambique tilapia (Oreochromis mossambicus). This technique involved extraction of predominantly cellular DNA from tilapia intestinal tracts and used a polymerase chain reaction assay to detect presence of type C1 toxin gene. We consistently detected C. botulinum type C cells in tilapia gastrointestinal contents at a level of 7.5 x 104 cells per 0.25 g material or 1.9 x 103 cells. This technique is useful for determining prevalence of the potentially active organisms within a given population of fish and may be adapted to other types of C. botulinum and vertebrate populations as well.

Prevalence of neurotoxic Clostridium botulinum type C in the gastrointestinal tracts of tilapia (Oreochromis mossambicus) in the Salton Sea.

Tilapia (Oreochromis mossambicus) have been implicated as the source of type C toxin in avian botulism outbreaks in pelicans (Pelecanus erythrorhynchos, Pelecanus occidentalis californicus) at the Salton Sea in southern California (USA). We collected sick, dead, and healthy fish from various sites throughout the Sea during the summers of 1999 through 2001 and tested them for the presence of Clostridium botulinum type C cells by polymerase chain reaction targeting the C(1) neurotoxin gene. Four of 96 (4%), 57 of 664 (9%), and five of 355 (1%) tilapia tested were positive for C. botulinum type C toxin gene in 1999, 2000, and 2001, respectively. The total number of positive fish was significantly greater in 2000 than in 2001 (P<0.0001). No difference in numbers of positives was detected between sick and dead fish compared with live fish. In 2000, no significant relationships were revealed among the variables studied, such as location and date of collection.

Preliminary evaluation of a simple in vitro test for the diagnosis of type C botulism in wild birds.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of type C botulinum toxin (Clostridium botulinum) in wild birds. This simple, antigen-capture ELISA utilizes polystyrene immunosticks as the solid substrate, chicken antitoxin (IgY) as the coating antibody, rabbit antitoxin as the primary antibody, and peroxidase-labeled goat-anti-rabbit as the secondary antibody. To evaluate the immunostick ELISA as a diagnostic test for avian botulism, known concentrations of toxin were added to heparinized blood collected from healthy birds and tested by both the ELISA and mouse bioassay. Also, blood samples from 236 bird carcasses submitted to the National Wildlife Health Center (NWHC) for cause of death determinations were tested by both procedures. Using < or = 0.5 ml as the test volume for both procedures, the ELISA was less sensitive, detecting 0.25 ng/ml of toxin compared to 0.12 ng/ml for the mouse bioassay. Using the same volume of test sample for diagnostic submissions (< or = 0.5 ml), the ELISA was positive for 60% of the 149 clinically-diagnosed cases of botulism, whereas the mouse bioassay was positive for 79%. However, we demonstrated that with larger sample volumes (> or = 1.0 ml), the sensitivity of the ELISA may be equivalent or better than the mouse test due to the concentrating effect of the ELISA procedure. These preliminary results suggest that when adequate sample volumes are available, the immunostick ELISA can replace the mouse test for the diagnosis of botulism in wild birds.

The inhibition of Clostridium botulinum type C by other bacteria in wetland sediments.

Bacteria with inhibitory activity against Clostridium botulinum type C were isolated from 32% of sediment samples (n = 1600) collected from 10 marshes in a northern California wetland over a 12 mo period. Aerobic and anaerobic bacteria with inhibitory activity were isolated from 12% and 23% of the samples, respectively. Bacteria with inhibitory activity were isolated from all 10 study sites and throughout the year. This study demonstrates that bacteria with inhibitory activity against C. botulinum type C occur naturally in wetland sediments.

Efficacy of a type C botulism vaccine in green-winged teal.

We tested the efficacy of a single dose of Botumink toxoid for protecting wild green-winged teal (Anas crecca) during botulism epizootics caused by Clostridium botulinum type C. We challenged control and immunized ducks with four different doses of type C botulinum toxin to determine the LD50 for this species and to evaluate vaccine protection. Fewer immunized ducks were affected with botulism than control ducks, indicating that a single dose of Botumink toxoid could increase the survival of ducks during epizootics. However, the frequency of immunized ducks with signs of botulism increased with the challenge dose of botulinum toxin. Even at doses of botulinum toxin approximately 2 to 4 green-winged teal LD50, about 50% of the immunized ducks were affected. We believe an improved vaccine or a better delivery system is required to justify immunization of wild birds for experimental survival studies.

Seasonal prevalence of Clostridium botulinum type C in sediments of a northern California wetland.

The prevalence of Clostridium botulinum type C (% of positive sediment samples) was determined in 10 marshes at Sacramento National Wildlife Refuge (SNWR), located in the Central Valley of California (USA), where avian botulism epizootics occur regularly. Fifty-two percent of 2,200 sediment samples collected over an 18-mo period contained C. botulinum type C (both neurotoxic and aneurotoxic) which was present throughout the year in all 10 marshes. The prevalence of C. botulinum type C was similar in marshes with either high or low botulism losses in the previous 5 yr. Marshes with avian botulism mortality during the study had similar prevalences as marshes with no mortality. However, the prevalence of C. botulinum type C was higher in marshes that remained flooded all year (permanent) compared with marshes that were drained in the spring and reflooded in the fall (seasonal). The prevalence of C. botulinum type C declined in seasonal marshes during the dry period. Similar declines did not occur in the permanently flooded marshes.

Desert bighorn sheep mortality due to presumptive type C botulism in California.

During a routine telemetry flight of the Mojave Desert (California, USA) in August 1995, mortality signals were detected from two of 12 radio-collared female desert bighorn sheep (Ovis canadensis) in the vicinity of Old Dad Peak in San Bernardino County (California). A series of field investigations determined that at least 45 bighorn sheep had died near two artificial water catchments (guzzlers), including 13 bighorn sheep which had presumably drowned in a guzzler tank. Samples from water contaminated by decomposing bighorn sheep carcasses and hemolyzed blood from a fresh bighorn sheep carcass were tested for the presence of pesticides, heavy metals, strychnine, blue-green algae, Clostridium botulinum toxin, ethylene glycol, nitrates, nitrites, sodium, and salts. Mouse bioassay and enzyme-linked immunosorbent assay detected type C botulinum toxin in the hemolyzed blood and in fly larvae and pupae. This, coupled with negative results from other analyses, led us to conclude that type C botulinum poisoning was most likely responsible for the mortality of bighorn sheep outside the guzzler tank.

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans).

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.

Prominent pancreatic endocrinopathy and altered control of food intake disrupt energy homeostasis in prion diseases.

Prion diseases are fatal neurodegenerative diseases that can induce endocrinopathies. The basis of altered endocrine function in prion diseases is not well understood, and the purpose of this study was to investigate the spatiotemporal relationship between energy homeostasis and prion infection in hamsters inoculated with either the 139H strain of scrapie agent, which induces preclinical weight gain, or the HY strain of transmissible mink encephalopathy (TME), which induces clinical weight loss. Temporal changes in body weight, feed, and water intake were measured as well as both non-fasted and fasted concentrations of serum glucose, insulin, glucagon, beta-ketones, and leptin. In 139H scrapie-infected hamsters, polydipsia, hyperphagia, non-fasted hyperinsulinemia with hyperglycemia, and fasted hyperleptinemia were found at preclinical stages and are consistent with an anabolic syndrome that has similarities to type II diabetes mellitus and/or metabolic syndrome X. In HY TME-infected hamsters, hypodipsia, hypersecretion of glucagon (in both non-fasted and fasted states), increased fasted beta-ketones, fasted hypoglycemia, and suppressed non-fasted leptin concentrations were found while feed intake was normal. These findings suggest a severe catabolic syndrome in HY TME infection mediated by chronic increases in glucagon secretion. In both models, alterations of pancreatic endocrine function were not associated with PrP(Sc) deposition in the pancreas. The results indicate that prominent endocrinopathy underlies alterations in body weight, pancreatic endocrine function, and intake of food. The prion-induced alterations of energy homeostasis in 139H scrapie- or HY TME-infected hamsters could occur within areas of the hypothalamus that control food satiety and/or within autonomic centers that provide neural outflow to the pancreas.

Oil and related toxicant effects on mallard immune defenses.

A crude oil, a petroleum distillate, and chemically dispersed oil were tested for their effects on resistance to bacterial infection and the immune response in waterfowl. Sublethal oral doses for mallards were determined for South Louisiana crude oil, Bunker C fuel oil, a dispersant--Corexit 9527, and oil/Corexit combinations by gizzard intubation. Resistance to bacterial challenge (Pasteurella multocida) was significantly lowered in mallards receiving 2.5 or 4.0 ml/kg of Bunker C fuel oil, 4.0 ml/kg of South Louisiana crude oil, and 4.0 ml/kg of a 50:1 Bunker C fuel oil/Corexit mixture daily for 28 days. Ingestion of oil or oil/Corexit mixtures had no effect on mallard antibody-producing capability as measured by the direct spleen plaque-forming assay.