Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses.

Comparative sequence analysis of six independent chicken and turkey parvovirus nonstructural (NS) genes revealed specific genomic regions with 100% nucleotide sequence identity. A polymerase chain reaction (PCR) assay with primers targeting these conserved genome sequences proved to be highly specific and sensitive to detecting parvoviruses in experimentally infected chickens. In a nationwide survey, a total of 138 field enteric samples from poultry flocks were tested by PCR for parvovirus presence. Of the tested chicken samples that were collected in 54 farms, 77% showed the presence of parvovirus, while 78% of the turkey samples that were received from 29 farms were parvovirus positive. For the first time, our data clearly demonstrate that parvoviruses are widely distributed in commercial poultry flocks in the United States. The high prevalence of parvovirus infection in birds from enteric disease-affected flocks suggests a potential role of these viruses in the etiology of enteric disease of poultry. Phylogenetic analyses comparing NS gene segments showed that most of the chicken and turkey parvovirus isolates formed separate phylogenetic groups. These findings suggest that the chicken and turkey parvoviruses might have diverged from a common ancestor and have subsequently undergone host-specific adaptation.

Development of an enzyme-linked immunosorbent assay to detect chicken parvovirus-specific antibodies.

Here we report the development and application of an enzyme-linked immunosorbent assay (ELISA) to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus vectors. In baculovirus recombinant-infected Sf9 cells, the chicken parvovirus (ChPV) structural viral protein 2 (VP2) was detected as an abundant protein, and the 60-kDa VP2 strongly reacted with parvovirus-infected chicken serum in Western blot. A semipurified VP2 was then used in capture ELISA. Sera from chickens experimentally infected with ChPV and sera from uninfected chickens were tested to evaluate the assay. The ELISA was 93.3% sensitive and 100% specific in detecting ChPV-infected birds. Subsequent assays identified IgG type ChPV-specific maternally acquired antibodies in day-old chickens and demonstrated the production of virus-specific antibodies in young birds following infection with ChPV. In our study, a specific antibody response of infected chickens was observed starting with IgM production between 14 and 21 days postinfection (DPI) and switching into a predominant IgG response by 32 DPI. The availability of an ELISA for detection of virus-specific antibodies and its ability to differentiate between maternally acquired antibodies and antibodies produced following acute infection could prove to be a valuable tool to characterize pathobiological properties and immunogenicity of ChPV.

Infection of mice with lyme disease spirochetes constitutively producing outer surface proteins a and B.

Outer surface protein A (OspA) of the Lyme disease spirochete is primarily produced in the tick vector. OspA, which is a receptor for attaching spirochetes to the tick gut, is down regulated as the spirochetes leave the tick and enter the mammalian host. Although OspA is not a major antigen produced in the mammal, the protein appears to be produced under some conditions and production has been linked to more severe disease. A Lyme disease vaccine based on recombinant OspA has been approved for human use. However, the vaccine is no longer available, in part because of fears that OspA causes arthritis in people. To further understand the consequences of OspA production in the host, we created a Borrelia burgdorferi mutant that was unable to down regulate OspA. C3H/HeN mice infected with this mutant developed a specific anti-OspA immune response, and the spirochetes were unable to persist in these mice. In contrast, immunodeficient SCID mice were persistently infected with the mutant. We conclude that spirochetes producing OspA and B from the flaB promoter in immunocompetent mice stimulate an immune response that clear the bacteria without any signs of disease development in the mice.

Plasmid requirements for infection of ticks by Borrelia burgdorferi.

Borrelia burgdorferi strain B31 MI commonly loses one or more of its complement of 21 extrachromosomal plasmids during normal handling procedures and during genetic manipulations. Certain plasmid losses cause an inability or reduction in the ability of spirochetes to infect mice. In the current study, nine strains of spirochetes with varying plasmid profiles were used to identify plasmids necessary for nymphal tick infection. Nymphal ticks were artificially fed the nine spirochete strains as well as the parental strain containing a full complement of plasmids. The capillary fed nymphs were allowed to feed on mice for at least 63 h and then examined for the presence of spirochetes in their guts and salivary glands. All spirochete strains tested were able to infect ticks guts, but to different degrees. We determined that the plasmids lp5, lp28-1, and cp9 were not required for infecting tick guts, whereas loss of lp25 and lp28-4 was associated with reduced gut infectivity. A reduction in the ability of spirochetes to invade salivary glands was seen in bacteria that did not have lp28-1, whereas cp9 was not required for salivary gland infection. This study has pinpointed specific plasmids whose absence is deleterious to infecting nymphal tick guts and salivary glands.

Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks.

The tick-borne bacterium Borrelia burgdorferi has over 20 different circular and linear plasmids. Some B. burgdorferi plasmids are readily lost during in vitro culture or genetic manipulation. Linear plasmid 25, which is often lost in laboratory strains, is required for the infection of mice. Strains missing linear plasmid 25 (lp25(-)) are able to infect mice if the BBE22 gene on lp25 is provided on a shuttle vector. In this study, we examined the role of lp25 and BBE22 in tick infections. We tested the hypothesis that complementation with BBE22 in spirochetes lacking lp25 would restore the ability of spirochetes to infect ticks. A natural tick infection cycle was performed by feeding larvae on mice injected with the parental, lp25(-), or lp25(-) BBE22-complemented spirochete strains. In addition, larvae and nymphs were artificially infected with different strains to study tick infections independent of mouse infections. B. burgdorferi missing lp25 was significantly impaired in its ability to infect larval and nymphal ticks. When an lp25(-) strain was complemented with BBE22, the ability to infect ticks was partially restored. Complementation with BBE22 allowed spirochetes lacking lp25 to establish short-term infections in ticks, but in most cases the infection prevalence was lower than that of the wild-type strain. In addition, the number of infected ticks decreased over time, suggesting that another gene(s) on lp25 is required for long-term persistence in ticks and completion of a natural infection cycle.

Reservoir competence of lesser mealworm (Coleoptera: Tenebrionidae) for Campylobacter jejuni (Campylobacterales: Campylobacteraceae).

The lesser mealworm, Alphitobius diaperinus (Panzer), is a carrier of Campylobacter spp. in poultry facilities; however, the beetle's importance in the epidemiology of campylobacteriosis is not known. A series of laboratory experiments were designed to test the vector and reservoir competence of the lesser mealworm for Campylobacter jejuni. In the first experiment, C. jejuni was swabbed onto the outer surface of adult and larval beetles to determine how long bacteria can survive on the beetles' exterior. Next, adult and larval mealworms were allowed to drink from a solution containing C. jejuni and the duration of internal carriage was monitored. For the third experiment, beetles drank from a Campylobacter suspension and the duration of fecal shedding of bacteria was determined. In the last experiment, 3-d-old chickens were fed either one or 10 infected beetles, and cloacal swabs were tested periodically for Campylobacter. C. jejuni was detected on the exterior of larval beetles for 12 h, from the interior of larvae for 72 h, and from the feces of larvae for 12 h after exposure. Ninety percent of the birds that consumed a single adult or larval beetles became Campylobacter-positive, whereas 100% of the birds that consumed 10 adults or larvae became positive. These experiments demonstrated that the lesser mealworm could acquire and harbor Campylobacter from an environmental source. We found that the lesser mealworm was capable of passing viable bacteria to chickens that consumed the beetle. The beetle should be included in attempts to maintain Campylobacter-free poultry facilities.