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.

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.

Emerging infectious diseases in wildlife.

The processes which give rise to emerging infectious diseases of wildlife can be categorised as follows: ecosystem alterations of anthropogenic or natural origin; movement of pathogens or vectors, via human or natural agency; and changes in microbes or in the recognition of emerging pathogens due to advances in the techniques of epidemiology. These are simplistic divisions because factors influencing the emergence of diseases of wild animals generally fall into more than one category. Mycoplasmosis among passerines is related to habitat changes and artificial feeding resulting in increased bird densities and subsequent disease transmission. The origin of this strain of Mycoplasma gallisepticum is not known. Hantavirus infections in rodents have emerged due to human-induced landscape alterations and/or climatic changes influencing population dynamics of hantavirus reservoir hosts, with disease consequences for humans. Movement of pathogens or vectors is a very important process by which diseases of wildlife expand geographic range. Although the origin of caliciviruses of rabbits and hares is somewhat obscure, their movement by humans, either deliberately or accidentally, has greatly expanded the distribution of these viruses. Rabies is an ancient disease, but geographic expansion has occurred by both natural and anthropogenic movements of wild animals. Human movement of amphibians may explain the distribution of the highly pathogenic chytrid fungus around the world. Newly recognised paramyxoviruses may reflect both changes in these pathogens and the development of techniques of identification and classification. Many more such examples of emerging diseases will arise in the future, given the extensive alterations in landscapes world-wide and movements of animals, vectors and pathogens. Those who study and diagnose diseases of wildlife must be alert for emerging diseases so that the impact of such diseases on wild animals, domestic animals and humans can be minimised.

Identification and localization of conserved antigenic epitopes on the G2 proteins of California serogroup Bunyaviruses.

California (CAL) serogroup Bunyaviruses are significant agents of arboviral encephalitis in humans. They are maintained and transmitted in nature by mosquitoes to preferred vertebrate amplifying hosts. The G2 envelope glycoprotein of La Crosse virus (LAC) was proposed by Ludwig et al. to be a determinant for virus attachment to mosquito midgut cells. Monoclonal antibodies to G2 neutralize the infectivity of pronase-treated virus for mosquito cells. We determined the location of antigenic sites on the LAC G2. We showed that antigenic areas present on the LAC G2 protein are conserved among viruses in the California encephalitis and Melao subgroups of the CAL serogroup, but not in trivatattus virus, nor within the BUN serogroup. A comparison of the G2 exodomain amino acid sequences of eight CAL and three BUN viruses with monoclonal antibodies (MAb) binding data predicted the possible location of the antigenic sites. We used in vitro mutagenesis of the LAC G2 gene to construct a set of G2 genes with replacement sequences in the coding regions for the suspected MAb binding sites. The native and mutated proteins were expressed in Hela cells and the ability of MAbs to bind to the expressed proteins was tested. Four discontinuous amino acid sequences, conserved among eight CAL serogroup viruses, were identified as contributing to two conformational binding domains for neutralizing LAC G2 MAbs.

Potential for evolution of California serogroup bunyaviruses by genome reassortment in Aedes albopictus.

Aedes albopictus was introduced into the United States in used tires in 1985. Its successful colonization of the upper Midwest has potential to alter the current epidemiology of bunyaviruses that circulate in the region. It is permissive for the replication of several arboviruses, including La Crosse (LACV) and Jamestown Canyon (JCV) bunyaviruses. In this study, we demonstrate the ability of LACV and JCV to coinfect Ae. albopictus mosquitoes and to form all six possible reassortant genotypes. All reassortant viruses infect Ae. albopictus orally and can be transmitted to suckling mice. All reassortants are neurovirulent in mice. However, reassortant viruses carrying the LACV M segment in the foreign genetic background of JCV are more neuroinvasive than JCV, or any other reassortant genotype. In addition, these reassortants can replicate in gerbils and infect Ae. triseriatus, characteristics of LACV, but not JCV. Because Ae. albopictus is spreading into new geographic areas and feeds on a variety of mammals, including humans, it has the potential to transmit new, emerging bunyaviruses in nature.

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.

La Crosse viremias in Mongolian gerbils (Meriones unguiculatis).

We examined the usefulness of mongolian gerbils (Meriones unguiculatus) as a new animal model for La Crosse virus (LACV) studies. Gerbils were exposed to LACV by either intramuscular (im) inoculation or exposure to transovarially infected Aedes triseriatus. Our studies indicate that gerbils may be a suitable animal model for LACV infection. Gerbils were susceptible to LACV, survived viral infection, and developed viremias and neutralizing antibody titers following exposure by im injection and by the bite of infected mosquitoes. Moreover, they are attractive to mosquito vectors. Gerbils have other advantages as laboratory vertebrate hosts for LACV; they are inexpensive, breed in captivity, and are usually mild-mannered and easy to handle. Thus, gerbils are a suitable model in the study of LACV pathogenesis as well as of transplacental and vector transmission.

Effects of La Crosse virus infection on pregnant domestic rabbits and mongolian gerbils.

The transplacental transmission of La Crosse virus (LACV) was evaluated in domestic rabbits (Oryctolagus cuniculus) and Mongolian gerbils (Meriones unguiculatis) as a potential mechanism for the maintenance of the virus. Rabbits were infected with LACV at different times of gestation by injection of viral suspensions or by exposure to LACV transovarially (TO) infected Aedes triseriatus. Pregnant gerbils were exposed between 16-24 days of gestation to LACV TO- infected Ae. triseriatus. Our results indicate that LACV can infect gerbils in utero. The LACV was isolated from the brain of suckling gerbils that died 3-5 days after birth from LACV-exposed mothers, representing the first evidence of LACV transplacental transmission. Microgliosis was found histologically in the cerebral cortex. In addition, LACV infection of both pregnant gerbils and rabbits resulted in in utero and neonatal mortality. La Crosse virus was not detected in surviving young of infected rabbits even after immunosuppression by administration of cyclophosphamide. Thus, there was no evidence of persistent infection of rabbits following in utero exposure. Surprisingly, some of the infected pregnant gerbils developed progressive paralysis 9-14-days postexposure, and LACV was isolated from the brains of these animals. Histopathologic studies of these tissue samples showed acute meningoencephalitis. The effects of natural LACV infection should be studied in pregnant amplifying hosts, such as chipmunks and squirrels, and in pregnant women.

Virulence of six strains of duck plague virus in eight waterfowl species.

Susceptibility of New World waterfowl to the Lake Andes strain of duck plague virus (DPV) was assessed by intramuscular inoculation of adult muscovies (Cairina moschata), mallards (Anas platyrhynchos), Canada geese (Branta canadensis), wood ducks (Aix sponsa), redheads (Aythya americana), gadwalls (Anas strepera), blue-winged teal (Anas discors), and pintails (Anas acuta). The relative virulence of DPV strains isolated from five United States and one Canadian location was established in muscovies, mallards, and Canada geese. Differences in DPV strain virulence were detected by formation of plaques in cell culture. Two strains that consistently formed plaques killed adult mallards while non-plaque forming strains killed hatchling but not adult mallards. Based on mortality after exposure to the Lake Andes strain, blue-winged teal, then wood ducks and redheads were highly susceptible, muscovies and gadwalls moderately susceptible, mallards and Canada geese less susceptible, and pintails the least susceptible. Mean death times were significantly (P < 0.01) different between adult muscovies (4.5 days) versus mallards and Canada geese (5.8 days each). Mean death time of the virulent Lake Andes and Minnesota strains were shorter (P < 0.05) than for the other four, less virulent DPV strains. Four of the less virulent strains killed hatchling but not adult mallards. Susceptibility to mortality was dependent upon age and route of inoculation. The intramuscular route of inoculation required the least amount of virus to kill mallard and muscovy ducks, the intranasal and conjunctival routes required more virus, and the oral route the most virus. This study was conducted from 1974 to 1977 between the months of September and April, with the exception of two titrations conducted in early May at the University of Wisconsin Department of Veterinary Science and the Charmany research facility of the University of Wisconsin-Madison.

La Crosse viremias in white-tailed deer and chipmunks exposed by injection or mosquito bite.

To further understand the role of wild mammals in the maintenance of La Crosse virus (LACV) in nature, we investigated the effects of inoculation method and virus source on the duration and amplitude of LACV viremia in vertebrate hosts. Earlier work suggested that deer are not sufficiently susceptible to LACV to play an important role in its maintenance. We re-evaluated the susceptibility of deer since subsequent studies showed that they constitute 65% of Aedes triseriatus blood meals, and thus would be exposed frequently to the virus. In our study, deer developed higher and longer viremia following exposure to LACV by infected Ae. triseriatus than those previously reported by inoculation with needle and syringe. However, susceptible Ae. triseriatus that fed on these viremic animals did not become infected. Because a large number of uninfected mosquitoes can feed upon a viremic deer in nature, we believe that deer should not be disregarded completely as a possible amplifier in the LACV transmission cycle. We also infected chipmunks to determine if there were significant differences in viremia response from mosquito delivery of virus to the chipmunk host, compared with artificial exposure by injection. Chipmunks exposed to infected mosquitoes had higher and longer viremias than the ones produced by intramuscular injection of an LACV suspension. These findings show the importance of using LACV infected mosquitoes for transmission experiments in mammals.

Serologic and virologic evidence of a Prospect Hill-like hantavirus in Wisconsin and Minnesota.

The purposes of this study were to determine if hantaviruses were present in the Great Lakes port areas of Wisconsin and Minnesota and if so, to identify which virus and which rodent host species were involved. Rodents were trapped in Duluth, Minnesota, Superior, Green Bay, and Milwaukee, Wisconsin, all ports of call for international maritime shipping. A total of 675 wild rodents were captured and tested, including 310 meadow voles (Microtus pennsylvanicus), 173 Norway rats (Rattus norvegicus), 179 Peromyscus spp., (including white footed mice [P. leucopus] and deer mice [P. maniculatus gracilis and P. maniculatus bairdii]), and 13 house mice (Mus musculus). Twenty percent of the rats, 17% of the meadow voles, 8% of the house mice, and 3% of the Peromyscus spp. had antibody to a hantavirus by immunofluorescent antibody assay (IFA). By the plaque-reduction neutralization test (PRNT), nine of 36 meadow voles, one of 4 P. leucopus, and one of 34 rats had hantavirus antibody, with the highest titers to Prospect Hill (PH) virus. All of the PRNT-seropositive individuals were from the twin cities of Superior and Duluth. Hantavirus antigen was detected in lung tissue by IFA in M. pennsylvanicus and Peromyscus spp., but not in rats. Two hantaviruses, designated SD-1 and SD-2, were isolated from M. pennsylvanicus captured in Duluth and found to be very similar to prototype PH virus by cross-IFA and cross-PRNT. Virus isolation attempts were unsuccessful from tissues of the Peromyscus spp. and the rats.

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.

Epidemiology of bluetongue viruses in the American tropics. Regional Bluetongue Team.

A study of the epidemiology of bluetongue viruses is in progress with the collaboration of 11 Central American and Caribbean countries. To date, over 200 bluetongue virus isolates have been obtained from cattle and sheep in sentinel groups distributed in the participating countries. Bluetongue serotypes identified include 1, 3, 6, and 12, virus types not previously recorded in the Western Hemisphere. Although the clinical impact of bluetongue virus infections in this hyperendemic environment appears to be minimal, the ubiquity of infection causes restrictions on the export of ruminant livestock and germ plasm. The stability of the Caribbean region ecosystem and the long-range implications of the interface with the northern temperate bluetongue virus ecosystem are reviewed.

Serological and microbial survey of Mycoplasma gallisepticum in wild turkeys (Meleagris gallopavo) from six western states.

From 1986 to 1989, sera from wild turkeys (Meleagris gallopavo), including three subspecies (M. gallopavo intermedia, M. gallopavo merriami and M. gallopavo mexicana) trapped in six western states were tested for antibody to Mycoplasma gallisepticum (MG) (n = 724), M. synoviae (MS) (n = 461) and M. meleagridis (MM) (n = 354) using the rapid plate agglutination (RPA) assay. Subsamples of these sera were also evaluated using the hemagglutination inhibition (HI) assay for antibody to MG (n = 664) and MS (n = 403). Attempts were made to isolate mycoplasmas by swabbing the trachea and cloaca of 190 live wild turkeys and from various tissues (sinus, nasal turbinates, trachea, lung, ovaries and oviduct) from 76 turkeys at necropsy. Isolates were identified using an immunobinding assay. Seroprevalence of MG, MS and MM in the RPA test was highly variable among years and geographic sites, ranging from 0 to 85%, 0 to 87%, and 0 to 83%, respectively, for each mycoplasma species. Of the 724 wild turkey sera tested, 200 (28%) were positive using the RPA assay, while only 20 (3%) of 664 sera tested using the HI assay were positive (at a titer greater than/= 1:80) for antibody to MG. Of the 461 sera tested 178 (39%) were RPA positive for MS, whereas none of the 403 samples tested by HI were positive for MS. Antibody to MM was detected in 72 (20%) of 354 turkey sera tested by RPA. Mycoplasmas were cultured from 81 (30%) of 266 wild turkeys, including 48 that were sampled live and 33 that were examined by necropsy. Mycoplasmas were isolated from every population in which culture was attempted. M. gallopavonis (MGP) was isolated from 37 (46%) of 81 birds which yielded mycoplasma, representing seven of 12 populations sampled. MG was isolated from lower respiratory tissues of one Rio Grande wild turkey trapped in Texas. M. synoviae was isolated from five of 16 Merriam's wild turkeys trapped in Arizona. Sera of birds from which MG or MS was isolated were positive to the respective antigen in the RPA test, but were negative by the HI assay. The RPA test was effective in identifying MG and MS infected turkeys despite lack of confirmation by the HI test. These data suggest that apparently healthy wild turkeys can carry pathogenic mycoplasmas and the currently used field test (RPA) can identify culture positive wild turkeys. Serological screening using the RPA test should be conducted on all wild turkeys prior to relocation.

Monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus.

Neutralizing monoclonal antibodies directed against the envelope glycoproteins of La Crosse virus (LACV) were prepared. Two antibodies immunoprecipitated the 120 kDa virus attachment protein for vertebrate cells, G1, while five immunoprecipitated the 35 kDa G2 protein, whose function is currently unknown. Two monoclonal antibodies were obtained that specifically precipitated both G1 and G2 from [35S]cysteine labeled LACV infected cell lysates. The G2 specific monoclonal antibodies had high neutralizing titers when assayed in mosquito cells but limited ability to neutralize virus in mammalian cells. The G1/G2 specific antibodies neutralized virus infectivity in both vertebrate and invertebrate cells at high titers. These results suggest that G2 is involved in the interaction of virus with mosquito cells and that G1 and G2 may share a common structural epitope relevant to their role as attachment proteins in vertebrate and mosquito cells. Monoclonal antibodies directed against G2 or G1/G2 have not previously been reported and should be useful tools for characterizing the biological functions of these molecules in the divergent micro-environments of vertebrate and invertebrate hosts.

Comparison of a modified Edward-type medium and a modified SP4-type medium for primary isolation of Mycoplasma gallisepticum (MG) from chickens vaccinated with the F strain of MG.

The efficacy of two media, an Edward-type medium (EPJ) and a modified SP4-type medium (SP4-PS), were compared for primary isolation of Mycoplasma gallisepticum (MG) from commercial layer chickens (n = 58) vaccinated with the live F strain of MG. Three groups of chickens that differed in the interval after vaccinal exposure to the F strain (32, 41, and 102 weeks) were studied at necropsy. Mycoplasma isolation was attempted from the trachea, sinus, and cloaca using lavage and swab techniques but was successful only from the trachea and sinus. MG was isolated from 39 (8.4%) of 463 culture attempts from 58 tracheal inocula and 58 sinus inocula. Isolation of MG was successful more frequently using EPJ medium than SP4-PS medium, and isolation occurred more often from the sinus than from the trachea. Of the 58 chickens studied, 19 (33%) were shown by culture to be infected with MG. Isolation was successful only from 32- and 41-week post-vaccination exposure groups. However, all chickens studied were serologically positive for MG antibody by rapid-plate agglutination and hemagglutination-inhibition assays.

Identification of F strain Mycoplasma gallisepticum isolates by detection of an immunoreactive protein.

Commercial laying hens were examined microbiologically at necropsy 31 or 42 weeks after aerosol vaccination with the F strain of Mycoplasma gallisepticum (MG). Mycoplasma isolates were studied in Western blots probed with polyclonal antiserum raised in rabbits to F strain immunogen. The persistence of the vaccine strain was demonstrated by detection of a 75-kilodalton immunoreactive protein, which was present in all MG isolates and thought to be a unique marker of the F strain. Use of PCA-F to probe Western blots allowed simultaneous identification of non-MG isolates, non-F strains of MG, and the F strain of MG.