ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE ADULT SPRINGBOK (ANTIDORCAS MARSUPIALIS), IMPALA (AEPYCEROS MELAMPUS), ALPACA (VICUGNA PACOS), AND RED-NECKED WALLABY (MACROPUS RUFOGRISEUS) OVER A YEAR.

Enterotoxemia is an important issue in various zoological taxa. In this study, serologic responses over a 1-yr period after vaccination with a multivalent clostridial vaccine were evaluated in 10 adult springboks (Antidorcas marsupialis), 12 impalas (Aepyceros melampus), seven alpacas (Vicugna pacos), and five red-necked wallabies (Macropus rufogriseus). Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay and determined as the percentage of inhibition (% inhib). Initial % inhib was (0.01-18.9)%. All animals received initial vaccination with a booster vaccine 4 weeks apart. Serum samples were collected at T0 (nonvaccinated), 15, 30, 60, 180, and 360 days postvaccination (dpv) for analysis. The vaccine induced a high antibody response that peaked at 15, 30, and 60 dpv in springboks, 30 and 60 dpv in impalas (P < 0.01), and 60 dpv in alpacas and wallabies (P < 0.01). The booster vaccine was followed by a high antibody response, which slowly decreased with time. The antibody response was significantly higher at 360 dpv than at T0 in wallabies and alpacas (P < 0.01). In impalas and springboks, it appeared that a booster every 6 mo might be required to maintain an antibody response above baseline (P < 0.01). Because no challenge studies were performed, it is unknown whether the measured humoral immune responses would have been protective. Further research is warranted to investigate protective effects of antibodies to inoculation challenge in nondomestic species.

PARAMETERS FOR IDENTIFYING FAILURE OF PASSIVE TRANSFER IN SITATUNGA (TRAGELAPHUS SPEKII).

Failure of passive transfer of immunity (FPT) leads to increased calf morbidity and mortality and requires intensive, time-sensitive, and often expensive management for nondomestic ruminants. Without species-specific information with which to make informed decisions, neonatal data from domestic ruminants are often extrapolated to nondomestic zoo-housed species. To date, there have been no studies evaluating FPT in sitatunga (Tragelaphus spekii). The goal of the present study was to establish parameters to characterize adequate passive transfer in sitatunga calves and compare them to published reference intervals in other species. Medical records of 22 sitatunga calves (12 female, 10 male) were reviewed. Seventeen of these calves were defined as "healthy," having survived at least 60 days without colostrum administration or a plasma transfusion. Calf weight, serum glucose, serum gamma-glutamyl transferase (GGT), total protein (TP), globulin concentrations, and results of a zinc sulfate turbidity test (ZSTT) were noted where possible. Mean birth weight of healthy calves at 24 hr was 4.5 kg (range: 3.76.5 kg, n = 12). The mean blood glucose in healthy calves was 152 mg/dl (range: 80-182, n = 16), mean serum TP concentration was 5.9 g/dl (range: 4.9-7.5, n = 16), mean serum globulin concentration was 3.3 g/dl (range: 1.7-4.7, n = 17), and mean serum GGT concentration was 466 U/L (range: 91-1901, n = 16). A ZSTT was performed for 10 healthy calves, resulting in four negative ZSTT results despite having no clinical signs of FPT and the calves having been observed nursing before testing. Sitatunga appear to have lower values for normal FPT parameters than those developed for domestic cattle. This study illustrates the difficulty of cross-species comparisons, as even closely related species can vary greatly in biologic parameters.

Seasonal patterns of hormones, macroparasites, and microparasites in wild African ungulates: the interplay among stress, reproduction, and disease.

Sex hormones, reproductive status, and pathogen load all affect stress. Together with stress, these factors can modulate the immune system and affect disease incidence. Thus, it is important to concurrently measure these factors, along with their seasonal fluctuations, to better understand their complex interactions. Using steroid hormone metabolites from fecal samples, we examined seasonal correlations among zebra and springbok stress, reproduction, gastrointestinal (GI) parasite infections, and anthrax infection signatures in zebra and springbok in Etosha National Park (ENP), Namibia, and found strong seasonal effects. Infection intensities of all three GI macroparasites examined (strongyle helminths, Strongyloides helminths, and Eimeria coccidia) were highest in the wet season, concurrent with the timing of anthrax outbreaks. Parasites also declined with increased acquired immune responses. We found hormonal evidence that both mares and ewes are overwhelmingly seasonal breeders in ENP, and that reproductive hormones are correlated with immunosuppression and higher susceptibility to GI parasite infections. Stress hormones largely peak in the dry season, particularly in zebra, when parasite infection intensities are lowest, and are most strongly correlated with host mid-gestation rather than with parasite infection intensity. Given the evidence that GI parasites can cause host pathology, immunomodulation, and immunosuppression, their persistence in ENP hosts without inducing chronic stress responses supports the hypothesis that hosts are tolerant of their parasites. Such tolerance would help to explain the ubiquity of these organisms in ENP herbivores, even in the face of their potential immunomodulatory trade-offs with anti-anthrax immunity.

Seroprevalence of bovine ephemeral fever virus in domesticated and wildlife species during epidemic and inter-epidemic periods (2000-2009) in Israel.

Bovine ephemeral fever (BEF) is an economically important vector-borne viral disease of cattle and buffalo. It has been reported from most of the world's tropical and subtropical regions. In the last few decades, outbreaks of BEF have occurred in Israel almost every other year. Several serological studies have demonstrated a wide range of wild animal species that are positive for BEF virus (BEFV) antibodies. However, the question of whether wild animals and domesticated species other than cattle also play an important role in the maintenance and transmission of BEFV in Israel remains. Here, we examined the prevalence of anti-BEFV antibodies in 942 samples collected from various wild, semi-captive and domesticated animal species during the years 2000-2009 using the serum neutralization (SN) method. SN test revealed the presence of BEFV-neutralizing antibodies in nine samples (0.96%), from three species: Bubalus bubalis (4/29, 13.79%), Gazella g. gazella (3/68, 4.44%) and Dama d. mesopotamica (2/296, 0.68%). All positive samples were collected in areas of earlier outbreaks. The low prevalence of positive animals and the solid correlation with prior outbreaks indicate that the tested species probably do not serve as virus reservoirs and may play only a minor role in the maintenance of BEFV in the Middle East.

Frequent and seasonally variable sublethal anthrax infections are accompanied by short-lived immunity in an endemic system.

Few studies have examined host-pathogen interactions in wildlife from an immunological perspective, particularly in the context of seasonal and longitudinal dynamics. In addition, though most ecological immunology studies employ serological antibody assays, endpoint titre determination is usually based on subjective criteria and needs to be made more objective. Despite the fact that anthrax is an ancient and emerging zoonotic infectious disease found world-wide, its natural ecology is not well understood. In particular, little is known about the adaptive immune responses of wild herbivore hosts against Bacillus anthracis. Working in the natural anthrax system of Etosha National Park, Namibia, we collected 154 serum samples from plains zebra (Equus quagga), 21 from springbok (Antidorcas marsupialis) and 45 from African elephants (Loxodonta africana) over 2-3 years, resampling individuals when possible for seasonal and longitudinal comparisons. We used enzyme-linked immunosorbent assays to measure anti-anthrax antibody titres and developed three increasingly conservative models to determine endpoint titres with more rigourous, objective mensuration. Between 52 and 87% of zebra, 0-15% of springbok and 3-52% of elephants had measurable anti-anthrax antibody titres, depending on the model used. While the ability of elephants and springbok to mount anti-anthrax adaptive immune responses is still equivocal, our results indicate that zebra in ENP often survive sublethal anthrax infections, encounter most B. anthracis in the wet season and can partially booster their immunity to B. anthracis. Thus, rather than being solely a lethal disease, anthrax often occurs as a sublethal infection in some susceptible hosts. Though we found that adaptive immunity to anthrax wanes rapidly, subsequent and frequent sublethal B. anthracis infections cause maturation of anti-anthrax immunity. By triggering host immune responses, these common sublethal infections may act as immunomodulators and affect population dynamics through indirect immunological and co-infection effects. In addition, with our three endpoint titre models, we introduce more mensuration rigour into serological antibody assays, even under the often-restrictive conditions that come with adapting laboratory immunology methods to wild systems. With these methods, we identified significantly more zebras responding immunologically to anthrax than have previous studies using less comprehensive titre analyses.

Evaluation of a commercial bovine colostrum replacer for achieving passive transfer of immunity in springbok calves (Antidorcas marsupialis).

Failure of passive transfer (FPT) occurs in ruminant species when there is inadequate absorption of immunoglobulins from the colostrum. In zoologic establishments, FPT can be a common occurrence in hand-raised ruminant neonates fed insufficient amounts of colostrum replacer (CR) and/or poor-quality CR. The goals of this study were to investigate the efficacy of a commercial bovine CR at achieving adequate passive transfer of immunity and evaluate tests to assess FPT in nondomestic ruminant species. In the past several years, research in dairy calves has shown that passive transfer rates can be dramatically improved if the CR dose is doubled. The treatment group (n = 10) consisted of springbok (Antidorcas marsupialis) calves fed Land O'Lakes bovine commercial CR at a dose of > or = 4.68 g of immunoglobulin G (22 g of CR powder) per kilogram of animal's body weight divided into five feedings over 24 hr. The control group (n=7) consisted of calves that nursed from their dams. This study reported no significant difference between the proportion of calves with adequate passive transfer in the treatment (80%) and control (71%) groups (P= 1.00). Morbidity and mortality rates until weaning were 0% in both groups. The study also determined the sensitivity and specificity values (n = 37) for five serum tests (y-glutamyl-transferase [GGT], globulin, glutaraldehyde coagulation [GC], sodium sulfite turbidity test, and total protein) used to determine passive transfer status in springbok calves. This study recommends the following serum tests and cutoff ranges for determining FPT in springbok calves: globulin < or = 1.85 mg/dl, GGT < or = 228.5 IU/ L, and GC > or = 28 min.

Evaluation of passive transfer in captive greater kudu (Tragelaphus strepsiceros).

Failure of passive transfer (FPT) in captive greater kudu (Tragelaphus strepsiceros) calves can lead to increased morbidity and mortality. In this retrospective study, serum samples from neonatal kudu calves were tested for immunoglobulin using different tests validated for domestic ruminants, including measurement of gamma globulin (GG) measured by protein electrophoresis, total solids (TS) measured by calibrated refractometry, total protein (TP) and globulins measured by colorimetry, gamma glutamyltransferase (GGT), and the zinc sulfate turbidity test (ZSTT). In a logistic regression model, TP, TS, globulins, and the natural log transform of GGT were the only significant parameters associated with FPT. Various historic parameters related to the dam, as well as calf weight, sex, glucose, and packed cell volume, were not significant. Based on the results, FPT in greater kudu is defined as GG of < 0.5 g/dl, a value lower than that in domestic cattle. TS measured by refractometry has an 80% sensitivity and a 100% specificity for FPT in greater kudu. With FPT defined as GG < 0.5 g/dl, kudu calves with a TS < 4.8 g/dl and a negative ZSTT have an increased probability of requiring medical intervention and additional diagnostics may be warranted.

Serologic responses of Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), wallaroos (Macropus robustus), and chimpanzees (Pan troglodytes) to an inactivated encephalomyocarditis virus vaccine.

Encephalomyocarditis virus (EMCV) is a picornavirus with a worldwide distribution, capable of infecting a wide range of species. Episodes of EMCV-associated mortality have been reported in zoos and national parks around the world, including sporadic cases at Taronga Zoo, Sydney. An inactivated EMCV vaccine was evaluated by inoculating Barbary sheep (Ammotragus lervia), Indian antelope (Antilope cervicapra), Eastern wallaroos (Macropus robustus), and chimpanzees (Pan troglodytes). A proportion of the vaccinated ungulates were administered a second vaccination 4 wk after the initial dose. Neutralizing antibody titers were monitored for a period of 12 mo. One month after vaccination, all vaccinated groups had developed significant antibody titers that persisted for at least 6 mo. Animals receiving two doses of vaccine had higher titers 3, 6, and 12 mo after the initial vaccination compared with animals receiving a single vaccine dose.

Study on the sequential tsetse-transmitted Trypanosoma congolense, T. brucei brucei and T. vivax infections to African buffalo, eland, waterbuck, N'Dama and Boran cattle.

Susceptibility of African buffalo, eland, waterbuck, N'Dama and Boran cattle to sequential Glossina morsitans centralis-transmitted infections of Trypanosoma congolense, T. brucei brucei and T. vivax was compared, and their possible role as reservoirs of these parasites for G. moristans centralis, G. pallidipes, G. austeni, G. brevipalpis and G. longipennis determined. The buffalo, eland, waterbuck and N'Dama controlled T. congolense parasitaemias and were able to prevent anaemia. By contrast, one Boran became severely anaemic whilst the other controlled parasitaemia and anaemia. When the above five species of Bovidae were rechallenged with T. brucei brucei they showed persistent parasitaemias but did not develop anaemia. The buffalo died of other causes. When the remaining four bovids were rechallenged with T. vivax they became infected with mixed T. vivax/T. b. brucei parasites. Eland, waterbuck and N'Dama controlled parasitaemias and anaemia whereas the Boran became anaemic. Cyclical development of T. congolense occurred in G. moristans centralis when fed on the bovid hosts, with buffalo being infective for tsetse flies for a much longer period. There was no relationship between the levels of T. congolense parasitaemia in the bovid hosts and the resultant infection rates in tsetse flies. Glossina m. centralis was more susceptible than G. pallidipes to T. brucei brucei whilst G. austeni the least; G. brevipalpis and G. longipennis were refractory to the mature infection. Again there was no relationship between T. brucei brucei parasitaemia levels in the hosts and infection rates in the flies. Glossina m. centralis and G. pallidipes showed mixed T. brucei brucei/T. vivax infections whilst G. austeni, G. brevipalpis and G. longipennis became infected with T. vivax alone. Tsetse flies showed higher T. vivax infection rates when fed on the hosts with high parasitaemias than thosewith low parasitaemias. Thus trypanotolerant African buffalo, eland, waterbuck, N'Dama as well as some trypanosusceptible Boran cattle can serve as reservoirs of single or mixed trypanosome infections for tsetse flies. This study has also shown that the Ag-ELISA on the sera from the five bovid hosts had low sensitivity and species-specificity. Examinations of thin wet blood films and buffy coats with a phase-contrast microscope were not sensitive enough to detect the parasites on all occasions. Xenodiagnosis using mice for T. brucei brucei and T. congolense infections, and tsetse flies for all the three trypanosome species were most sensitive for the detection of trypanosome infections in the bovid hosts.

Morphological evidence for infection of impala, Aepyceros melampus, platelets by a rickettsia-like organism.

Ultrastructural studies revealed the presence of a parasite, believed to be Ehrlichia platys, in the blood platelets of impala. At the time of blood sampling all the animals appeared healthy. This is the first report on the presence of this rickettsia in these animals, previously described in canine platelets.

Cross-reactivity of workshop antibodies with cells from domestic and wild ruminants.

Reactivities of the monoclonal antibodies (mAbs) from the workshop panel with cells from cattle, sheep, goats, Cape buffalo (Syncerus caffer) and waterbuck (Kobus defassa) were tested. One hundred and sixty-nine mAbs reacted with bovine cells and 111 with sheep cells; 86 were shown to react with goat cells, 71 with buffalo cells and 70 with waterbuck cells. Some mAbs cross-reacted with all five ruminants tested, and are likely to react with epitopes that are conserved in other ruminant species. Such mAbs will obviate the need to produce mAbs panels to leukocyte antigens of other ruminants.

Characterisation of lymphocyte populations in African buffalo (Syncerus caffer) and waterbuck (Kobus defassa) with workshop monoclonal antibodies.

In order to measure different lymphocyte populations in buffalo (Syncerus caffer) and waterbuck (Kobus defassa), we analysed the monoclonal antibodies from the 1st International Workshop on Leukocyte Antigens in Cattle, Sheep and Goats for suitable cross-reactive reagents. Peripheral blood mononuclear cells from three buffalo and three waterbuck were tested with the whole panel of monoclonal antibodies (mAbs) together with some additional antibodies against MHC and Ig. In some clusters almost all antibodies cross-reacted (CD2, CD8), in others almost none cross-reacted (CD4, CD5) and in cluster CD6, mAbs only reacted with buffalo but not waterbuck. Double staining experiments were performed on buffalo PBM with the cross-reacting antibodies, to confirm that they detected similar cell populations as in bovine PBM. This was shown with reagents against CD2, CD4, CD6, CD8, CD11, WC1, WC3 and Ig. The molecular weights of the buffalo antigens correlated well with those of the homologous cattle antigens. In the CD5 cluster, only one mAb reacted with the two wild species, and defined an unusual CD2+ CD5- cell population in buffalo. Also mAbs cross-reacting with buffalo MHC class II detected unusual expression on resting T cells. From the results presented, it is clear that the workshop panel contains mAbs against the most important T and B cell antigens of buffalo and probably waterbuck, which will allow us to compare functional lymphocyte populations in cattle and wild ruminants.

Antibodies to some pathogenic agents in free-living wild species in Tanzania.

A total of 535 sera from eight species of wildlife were collected from different game areas in Tanzania between 1987 and 1989. These sera were tested for antibodies against foot-and-mouth disease, bovine herpes virus types 1 and 2, lumpy skin disease, bovine viral diarrhoea, Akabane, bovine ephemeral fever, bluetongue, enzootic bovine leucosis, African horse sickness and African swine fever viruses and Brucella abortus based on the expected species susceptibility. Sera from buffalo Syncerus caffer, wildebeest Connochaetes taurinus and topi Damaliscus korrigum contained antibodies against the majority of the pathogens tested. Antibodies to fewer pathogens were detected in sera from the other species. No antibodies to lumpy skin disease virus were detected in any of the sera examined. African horse sickness antibodies were detected in sera from Zebra and African swine fever antibodies were detected in wart hog. The occurrence of antibodies to these agents suggests that wild species act as reservoirs of infection for some of these pathogens. However, until the susceptibility of individual species is proven by isolation of the aetiological agents their role must remain speculative.

Immunoblotting analysis of the reaction of wildebeest, sheep and cattle sera with the structural antigens of alcelaphine herpesvirus-1 (malignant catarrhal fever virus).

Malignant catarrhal fever (MCF) is a disease of cattle and some other ruminants caused by alcelaphine herpesvirus-1 (AHV-1), a virus of wildebeest. The disease also occurs in the absence of wildebeest and is then thought to be caused by a viral agent harboured by the sheep. The structural proteins of AHV-1 have been used as antigens for the immunoblotting analysis of sera from wildebeest, sheep and cattle infected by either AHV-1 or the "sheep-associated" form of the disease. Wildebeest sera showed a uniform response reacting strongly with six polypeptides. Sheep sera also gave positive results but individual sera reacted with varying subsets of the antigens recognized by wildebeest. These results support the earlier suggestion that sheep harbour a herpesvirus related to AHV-1. A bovine serum from a case of MCF caused by AHV-1 also reacted only with a subset of the six wildebeest-reactive polypeptides. Sera from cattle affected with the "sheep-associated" form of the disease gave reactions in only two of the eight cases tested; both positive sera reacted to a few polypeptides only.

Isolation of Legionella pneumophila from calves and the prevalence of antibodies in cattle, sheep, horses, antelopes, buffaloes and rabbits.

The lungs of 139 calves presented for autopsy and 29 healthy slaughtered calves were examined for Legionella by culture and by direct immunofluorescence (DIF) with fluorescein-conjugated antisera. About 17% of the cadaver lungs and 4% of lungs from slaughtered animals were positive by DIF. Legionella organisms were only isolated from the lungs of two cadavers (L. pneumophila, serogroup 1). In a prevalence study of antibodies to Legionella in domestic and wild animals of various species, titers of greater than or equal to 64 were demonstrated by indirect immunofluorescence in sera of 10% of dairy cattle, 5% of beef cattle, 4% of sheep, 22% of antelopes, 35% of horses, 36% of buffaloes and 0% of laboratory rabbits. The isolation of Legionella from lung tissue is evidence for a possible etiologic role of Legionella spp. in natural pathology of animals.

Antibodies to bovine bacterial and viral pathogens in pronghorns in Alberta, 1983.

Sera from 210 pronghorns (Antilocapra americana) ranging in southeastern Alberta were tested for antibodies to disease agents present in indigenous cattle. No antibodies to Brucella abortus, Leptospira interrogans serovars pomona, hardjo, or grippotyphosa, or infectious bovine rhinotracheitis virus were found. Antibodies at prevalences of 43.8% and 49.2% were detected to bovine virus diarrhea (BVD) and parainfluenza type 3 (PI-3) viruses, respectively. The much higher prevalence of BVD virus antibodies in cattle than in pronghorns, and the occurrence of clinical bovine PI-3 infection in the study area, suggest that cattle may be a source of infection to the pronghorns.

Immune effector mechanisms involved in the control of parasitaemia in Trypanosoma brucei-infected wildebeest (Connochaetes taurinus).

The course of Trypanosoma brucei infection in the wildebeest (Connochaetes taurinus) was studied. A low but persistent parasitaemia developed in all five wildebeest following intravenous inoculation with 1 X 10(8) organisms of clone ILTat 2.1. Unlike cattle controls, however, the wildebeest did not develop anaemia. In both cattle and wildebeest, radioimmunoassay studies revealed a classical sequence of production of IgM, IgG1 and IgG2 antibodies which had the capacity to bind to the corresponding purified variable surface glycoprotein and to neutralize the infectivity of ILTat 2.1. Investigations into the interaction between post-infection sera, trypanosomes and freshly isolated peripheral blood leucocytes (PBL) of wildebeest and cattle showed that sera from the wildebeest had a higher capacity to induce adherence of trypanosomes to homologous PBL. The adherence and phagocytosis-inducing activity resided in the IgM fraction. Cross-testing of the antibodies and PBL revealed that wildebeest IgM antibodies induced high adherence indices when tested on cattle PBL. High adherence indices were also observed when cattle IgM antibodies were tested on PBL of wildebeest. It was concluded that the phagocytic system of the wildebeest was superior to that of cattle, that freshly prepared wildebeest PBL bear receptors for wildebeest as well as cattle IgM, and that cattle PBL bear a receptor for wildebeest IgM that would appear to be different from that for cattle IgM.

Natural agglutinins to African trypanosomes.

Agglutinins to uncoated culture forms of the African trypanosomes, T. congolense and T.b. brucei were detected in sera from a variety of mammals not exposed to the parasites. The agglutinins in bovine serum were shown to be specific antibodies with opsonic properties selective for the species of trypanosome. These findings suggest a possible role for the glycoprotein coat in preventing access of cross-reacting antibodies to the plasma membrane of African trypanosomes.

Antibodies in carrier wildebeest to the lymphoproliferative herpesvirus of malignant catarrhal fever.

Six types of antibody to malignant catarrhal fever virus (MCFV) were measured in 132 sera collected from Wildebeest in Kenya Masailand. The titre of all types of antibody declined slowly with increasing age of the wildebeest. A significantly greater proportion of wildebeest calves had higher titres of antibodies to MCFV early antigens, IgM antibodies to MCFV late antigens and complement-fixing antibodies, than did older animals. One seronegative calf, reared in isolation without colostrum, became seropositive 4 1/2 weeks after birth but did not show any clinical signs indicative of MCFV infection. Similarities between MCFV infection of wildebeest calves and other inapparent infections with lymphoproliferative herpesviruses are discussed.