Lalitha Peddireddi

Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis

Ehrlichia chaffeensis and Ehrlichia canis are tick‐transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell‐dependent. The differentially expressed proteins included those from the p28/30‐Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28‐Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30‐10 protein, an orthologue of the E. chaffeensis p28‐Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post‐translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30‐Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non‐vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30‐Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.

Differential Clearance and Immune Responses to Tick Cell-Derived versus Macrophage Culture-Derived Ehrlichia chaffeensis in Mice

ABSTRACT Human monocytic ehrlichiosis is caused by a tick-transmitted rickettsia, Ehrlichia chaffeensis. We recently reported that E. chaffeensis grown in tick cells expresses different proteins than bacteria grown in macrophages. Therefore, we tested the hypothesis that immune responses against E. chaffeensis would be different if the mice are challenged with bacteria grown in macrophages or tick cells. We assessed the E. chaffeensis clearance from the peritoneum, spleen, and liver by C57BL/6J mice using a TaqMan-based real-time reverse transcription-PCR assay. Macrophage-grown E. chaffeensis was cleared in 2 weeks from the peritoneum, whereas the pathogen from tick cells persisted for nine additional days and included three relapses of increasing bacterial load separated by three-day intervals. Tick cell-grown bacteria also persisted in the livers and spleens with higher bacterial loads compared to macrophage-grown bacteria and fluctuated over a period of 35 days. Three-day periodic cycles were detected in T-cell CD62L/CD44 ratios in the spleen and bone marrow in response to infections with both tick cell- and macrophage-grown bacteria and were accompanied by similar periodic cycles of spleen cell cytokine secretions and nitric oxide and interleukin-6 by peritoneal macrophages. The E. chaffeensis-specific immunoglobulin G response was considerably higher and steadily increased in mice infected with the tick cell-derived E. chaffeensis compared to DH82-grown bacteria. In addition, antigens detected by the immunoglobulins were significantly different between mice infected with the E. chaffeensis originating from tick cells or macrophages. The differences in the immune response to tick cell-grown bacteria compared to macrophage-grown bacteria reflected a delay in the shift of gene expression from the tick cell-specific Omp 14 gene to the macrophage-specific Omp 19 gene. These data suggest that the host response to E. chaffeensis depends on the source of the bacteria and that this experimental model requires the most natural inoculum possible to allow for a realistic understanding of host resistance.

Molecular characterization of Ehrlichia interactions with tick cells and macrophages.

Several tick-transmitted Anaplasmataceae family rickettsiales of the genera Ehrlichia and Anaplasma have been discovered in recent years. Some species are classified as pathogens causing emerging diseases with growing health concern for people. They include human monocytic ehrlichiosis, human granulocytic ewingii ehrlichiosis and human granulocytic anaplasmosis which are caused by Ehrlichia chaffeensis, E. ewingii and Anaplasma phagocytophilum, respectively. Despite the complex cellular environments and defense systems of arthropod and vertebrate hosts, rickettsials have evolved strategies to evade host clearance and persist in both vertebrate and tick host environments. For example, E. chaffeensis growing in vertebrate macrophages has distinct patterns of global host cell-specific protein expression and differs considerably in morphology compared with its growth in tick cells. Immunological studies suggest that host cell-specific differences in Ehrlichia gene expression aid the pathogen, extending its survival. Bacteria from tick cells persist longer when injected into mice compared with mammalian macrophage-grown bacteria, and the host response is also significantly different. This review presents the current understanding of tick-Ehrlichia interactions and implications for future.

Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs.

We determined tissue localization, shedding patterns, virus carriage, antibody response, and aerosol transmission of Porcine epidemic diarrhea virus (PEDV) following inoculation of 4-week-old feeder pigs. Thirty-three pigs were randomly assigned to 1 of 3 groups for the 42-day study: inoculated (group A; n = 23), contact transmission (group B; n = 5), and aerosol transmission (group C; n = 5). Contact transmission occurred rapidly to group B pigs whereas productive aerosol transmission failed to occur to group C pigs. Emesis was the first clinical sign noted at 3 days postinoculation (dpi) followed by mild to moderate diarrhea lasting 5 more days. Real-time PCR detected PEDV in fecal and nasal swabs, oral fluids, serum, and gastrointestinal and lymphoid tissues. Shedding occurred primarily during the first 2 weeks postinoculation, peaking at 5–6 dpi; however, some pigs had PEDV nucleic acid detected in swabs collected at 21 and 28 dpi. Antibody titers were measurable between 14 and 42 dpi. Although feces and intestines collected at 42 dpi were PEDV negative by PCR and immunohistochemistry, respectively, small intestines from 70% of group A pigs were PCR positive. Although disease was relatively mild and transient in this age group, the results demonstrate that 4-week-old pigs are productively infected and can sustain virus replication for several weeks. Long-term shedding of PEDV in subclinically affected pigs should be considered an important source for PEDV transmission.

Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes.

BackgroundEhrlichia chaffeensis is a rickettsial agent responsible for an emerging tick-borne illness, human monocytic ehrlichiosis. Recently, we reported that E. chaffeensis protein expression is influenced by macrophage and tick cell environments. We also demonstrated that host response differs considerably for macrophage and tick cell-derived bacteria with delayed clearance of the pathogen originating from tick cells.ResultsIn this study, we mapped differences in the promoter regions of two genes of p28-Omp locus, genes 14 and 19, whose expression is influenced by macrophage and tick cell environments. Primer extension and quantitative RT-PCR analysis were performed to map transcription start sites and to demonstrate that E. chaffeensis regulates transcription in a host cell-specific manner. Promoter regions of genes 14 and 19 were evaluated to map differences in gene expression and to locate RNA polymerase binding sites.ConclusionRNA analysis and promoter deletion analysis aided in identifying differences in transcription, DNA sequences that influenced promoter activity and RNA polymerase binding regions. This is the first description of a transcriptional machinery of E. chaffeensis. In the absence of available genetic manipulation systems, the promoter analysis described in this study can serve as a novel molecular tool for mapping the molecular basis for gene expression differences in E. chaffeensis and other related pathogens belonging to the Anaplasmataceae family.

Pooling of cultured samples and comparison of multistate laboratory workflows with the MagMAX sample preparation system and VetMAX quantitative polymerase chain reaction reagents for detection of Tritrichomonas foetus–colonized bulls

The objectives of the current study were 1) to compare sample preparation workflows and quantitative real-time polymerase chain reaction assays (qPCR) as currently used in veterinary diagnostic laboratories with a study protocol utilizing commercially available reagents for individual Tritrichomonas foetus testing, 2) to assess the accuracy of pooling cultured smegma samples followed by extraction and qPCR testing as used in the study laboratory, and 3) to assess the specificity of the currently used primers and probes by sequencing all positive and presumptive positive samples identified in the study laboratory in an attempt to capture any nucleotide variability between T. foetus isolates and to rule out false-positive results possibly due to Simplicimonas moskowitzi. Eight hundred three cultured smegma samples were collected from different regions of the United States with the collaboration of 5 veterinary testing laboratories. The samples were processed individually by the respective laboratories, and then sent to the study laboratory and retested using the study protocol. Comparison testing showed an overall agreement of 95.89% between the veterinary testing laboratories and the study laboratory. One hundred seventy-six positive or presumptive positive samples plus 625 negative qPCR samples were combined and retested using a pooling protocol. Pools consisted of 1 positive sample and 4 negative samples (1/5). These pools were processed using the same study laboratory protocols, and 96% of the positive samples were detected in these pools. Nested PCR followed by sequencing confirmed 175 of the 178 samples classified as positive or presumptive positive in the study laboratory as containing T. foetus–specific DNA.

Evaluation of conjunctival bacterial flora in a herd of goats in the Midwestern United States

OBJECTIVE To identify conjunctival bacterial flora in healthy adult and newborn kid goats in the Midwestern United States and to compare vaginal and ocular surface flora in dam-kid pairs. ANIMALS STUDIED Thirty mixed-breed (crosses between Boer, Kiko, and Syfan Spanish) goats, 20 adult does and 10 newborn kids. PROCEDURES One eye of 15 adult goats (n = 15 eyes) and 5 kids (n = 5 eyes) was randomly selected. A subset of 5 adults (n = 10 eyes) and 5 kids (n = 10 eyes) underwent bilateral sampling. Each recently kidded dams vaginal canal (n = 10) was also sampled. Two swabs were collected from each sample site for aerobic bacterial culture and Mycoplasma and Chlamydia spp. PCR. RESULTS Of the animals with positive cultures, Staphylococcus and Streptococcus were the most common bacterial genera from the conjunctival sac of adult (16/17; 94%) and kid (5/5; 100%) goats; three adults and 5 kids had no growth of bacteria on aerobic culture. Moraxella bovoculi was the most common single bacteria in adults, in 9 eyes (36%) of 8 animals (40%). Staphylococcus equorum was identified in all 5 kids with positive cultures. Mycoplasma sp. DNA was detected in 7 animals. Chlamydia sp. DNA was not detected in any sample. Four of 10 dam-kid pairs had identical bacteria isolated from the dams vaginal sample and the kids conjunctival sample. CONCLUSIONS Staphylococcus and Streptococcus were the most common conjunctival bacterial genera in this goatherd. Moraxella bovoculi was the most common single bacteria isolated from adults, and Staphylococcus equorum was the most common bacteria in kids. Mycoplasma sp. occurred infrequently at the ocular surface of adult and kid goats. A convincing association between dam-kid vaginal-conjunctival samples was not identified.

A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

Abstract Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R2) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

Isolation and characterization of Ehrlichia chaffeensis RNA polymerase and its use in evaluating p28 outer membrane protein gene promoters

BackgroundEhrlichia chaffeensis is a tick-transmitted rickettsial pathogen responsible for an important emerging disease, human monocytic ehrlichiosis. To date how E. chaffeensis and many related tick-borne rickettsial pathogens adapt and persist in vertebrate and tick hosts remain largely unknown. In recent studies, we demonstrated significant host-specific differences in protein expression in E. chaffeensis originating from its tick and vertebrate host cells. The adaptive response of the pathogen to different host environments entails switch of gene expression regulated at the level of transcription, possibly by altering RNA polymerase activity.ResultsIn an effort to understand the molecular basis of pathogen gene expression differences, we isolated native E. chaffeensis RNA polymerase using a heparin-agarose purification method and developed an in vitro transcription system to map promoter regions of two differentially expressed genes of the p28 outer membrane protein locus, p28-Omp14 and p28-Omp19. We also prepared a recombinant protein of E. chaffeensis σ70 homologue and used it for in vitro promoter analysis studies. The possible role of one or more proteins presents in E. chaffeensis lysates in binding to the promoter segments and on the modulation of in vitro transcription was also assessed.ConclusionsOur experiments demonstrated that both the native and recombinant proteins are functional and have similar enzyme properties in driving the transcription from E. chaffeensis promoters. This is the first report of the functional characterization of E. chaffeensis RNA polymerase and in vitro mapping of the pathogen promoters using the enzyme. This study marks the beginning to broadly characterize the mechanisms controlling the transcription by Anaplasmataceae pathogens.