Immunological responses in the mouse host to a cloned antigen of Taeniacrassiceps

Adult female Swiss-Webster mice were immunized either intraperitoneally (IP) or subcutaneously (SQ) with cyst fluid or a genetically engineered fusion protein, Taenia carassiceps antigen 2-maltose binding protein (TCA2-MBP) from Taenia crassiceps metacestodes, or with live, non-budding cysts SQ, and then challenged IP with T. crassiceps metacestodes and necropsied 9 weeks later. Numbers of peripheral blood eosinophils were increased after IP immunization, but were not increased after SQ immunization or with SQ cysts given before the challenge infection. Eosinophil numbers gradually decreased over the course of the experiment, and were not found in increased numbers in the blood or peritoneal cavity at necropsy. Antigen-specific antibody responses were seen at day 14 or 28 in IP and SQ immunized groups; IgG3 and IgG3 isotypes continued to increase over the course of the experiment. A significant protective response was induced by immunization with the cyst fluid (15 + 4, X + SE recovered larvae) or the TCA2-MBP (22+12) given IP, but not SQ (122+36l ; 207+53, respectively) as measured by the numbers of larvae recovered at necropsy. Live cysts given SQ resulted in reduced numbers of cysts in the peritoneal cavity (188+66), but was not as effective as cyst fluid or TCA2-MBP given IP. Locally (IP) induced immune responses may be involved in the development of the protective response to a challenge infection with T. crassiceps metacestodes

A multiplex polymerase chain reaction assay to simultaneously distinguish Cryptosporidium species of veterinary and public health concern in cattle

Four species of Cryptosporidium are routinely found in cattle: Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium ryanae, and Cryptosporidium andersoni. It is important to determine the species of Cryptosporidium in infected cattle because C. parvum is the only serious pathogen for humans as well as cattle. Identification of Cryptosporidium species and genotypes currently relies on molecular methods such as polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or gene sequencing. Incorporation of these techniques in a routine veterinary diagnostic laboratory is cost prohibitive. As such, their applications are limited primarily to research and a few public health laboratories. To overcome this problem, a multiplex PCR assay was developed for simultaneously detecting the 4 species of Cryptosporidium that commonly infect cattle. This assay specifically identifies Cryptosporidium oocysts present in cattle feces, improves the detection of mixed infections, reduces the time and cost relative to current sequencing methods, and further demonstrates the shortcomings of sequencing as the definitive method for identification when analyzing samples containing mixed infections

PCR as a diagnostic and quantitative technique in veterinary parasitology

Over the past 15 years, there has been a dramatic evolution in molecular approaches to study parasites and parasitic diseases. Many of these advancements have been brought about through the development of new applications of the polymerase chain reaction (PCR). Enhancements in sensitivity that can be achieved using PCR now permit scientists to investigate changes at the level of a single cell, far below what is often needed for parasite-derived applications. PCR has had a substantial impact on advances made in the areas of parasite systematics and epidemiology, immunology and host–parasite interactions, recombinant DNA vaccine development and most re- cently, the analysis of whole genomes either through directly sequencing the DNA, the analysis of expressed sequence tags (ESTs) or through the rapidly growing field of functional genomics. This paper, however, focuses on the application of PCR methodology to parasite detection and differen- tiation, and the diagnosis of disease. Specific attention is given to advances provided by multiplex PCR, fluorescence-based “real-time” PCR, and the utilization of PCR as a quantitative technique. Published by Elsevier Science B.V

Immunological responses in the mouse host to a cloned antigen of Taeniacrassiceps

Adult female Swiss-Webster mice were immunized either intraperitoneally (IP) or subcutaneously (SQ) with cyst fluid or a genetically engineered fusion protein, Taenia carassiceps antigen 2-maltose binding protein (TCA2-MBP) from Taenia crassiceps metacestodes, or with live, non-budding cysts SQ, and then challenged IP with T. crassiceps metacestodes and necropsied 9 weeks later. Numbers of peripheral blood eosinophils were increased after IP immunization, but were not increased after SQ immunization or with SQ cysts given before the challenge infection. Eosinophil numbers gradually decreased over the course of the experiment, and were not found in increased numbers in the blood or peritoneal cavity at necropsy. Antigen-specific antibody responses were seen at day 14 or 28 in IP and SQ immunized groups; IgG3 and IgG3 isotypes continued to increase over the course of the experiment. A significant protective response was induced by immunization with the cyst fluid (15 + 4, X + SE recovered larvae) or the TCA2-MBP (22+12) given IP, but not SQ (122+36l ; 207+53, respectively) as measured by the numbers of larvae recovered at necropsy. Live cysts given SQ resulted in reduced numbers of cysts in the peritoneal cavity (188+66), but was not as effective as cyst fluid or TCA2-MBP given IP. Locally (IP) induced immune responses may be involved in the development of the protective response to a challenge infection with T. crassiceps metacestodes

Transmissible gastroenteritis virus: Identification of M protein-binding peptide ligands with antiviral and diagnostic potential

The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p \u3c 0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment

A novel algorithm to define infection tendencies in H1N1 cases in Mainland China

Incidences of H1N1 viral infections in Mainland China are collected by the Ministry of Health, the People’s Republic of China. The number of confirmed cases and the timing of these outbreaks from May 13 to July 22, 2009 were obtained and subjected to a novel mathematical model to simulate the infection profile (time vs number). The model was predicated upon the grey prediction theory which allows assignment of future trends using limited numbers of data points. During the period of our analysis, the number of confirmed H1N1 cases in Mainland China increased from 1 to 1772. The efficiency of our model to simulate these data points was evaluated using Sum of squares of error (SSE), Relative standard error (RSE), Mean absolute deviation (MAD) and Average relative error (ARE). Results from these analyses were compared to similar calculations based upon the grey prediction algorithm. Using our equation, defined herein as equation D–R, results showed that SSE = 6742.00, RSE = 10.69, MAD = 7.07, ARE = 2.47% were all consistent with the D–R algorithm performing well in the estimation of future trends of H1N1 cases in Mainland China. Calculations using the grey theory had no predictive value [ARE for GM(1,1) = 104.63%]. To validate this algorithm, we performed a second analysis using new data obtained from cases reported to the WHO and CDC in the US between April 26 and June 8, 2009. In like manner, the model was equally predictive. The success of the D–R mathematical model suggests that it may have broader application to other viral infections among the human population in China and may be modified for application to other regions of the worl

A multiplex PCR assay for differentiating economically important gastrointestinal nematodes of cattle

A multiplex polymerase chain reaction (PCR) test was developed for identifying gastrointestinal (GI) nematodes that commonly infect cattle. This assay was developed using adult-derived genomic DNA and shown capable of discriminating parasite eggs from the feces of experimentally-infected animals at both the species and genus levels. Sequence data from internal (ITS) and external (ETS) transcribed spacers of the ribosomal DNA (rDNA) repeats as well as the 3′-end of the small subunit rDNA and 5′-end of the large subunit rDNA were used to generate five primer sets which, when used simultaneously in a multiplex PCR, produce a unique electrophoretic DNA banding pattern charac- terized by a single DNA fragment for Ostertagia ostertagi (257 bp), Haemonchus placei (176 bp),Oesophagostomum radiatum (329 bp), Trichostrongylus colubriformis (243 bp) and Cooperia on- cophora (151 bp). In a similar manner, the constructed primer sets amplified DNA from Ostertagia lyrata, Haemonchus contortus, Trichostrongylus axei, Cooperia surnabada and Cooperia punctata. With respect to H. contortus, a closely migrating doublet was generated suggesting size heterogene- ity in the ETS which is consistent with multiple rDNA repeat units within this species. PCR analyses using mixtures of monospecifically-purified nematode eggs indicated a sensitivity of less than 0.5 egg-DNA equivalent per species. Although, not designed as a quantitative technique, relative PCR signal intensities corresponded to relative egg burdens within the DNA samples from mixed species of eggs. Published by Elsevier Science B.V

Wild ruminants as reservoirs of domestic livestock gastrointestinal T nematodes

Gastrointestinal nematode (GIN) infections in cattle cause appetite suppression which leads to poor feed conversion, reduced weight gain and reduced milk production. Overuse and exclusive reliance on anthelmintic drugs has resulted in widespread resistance in many parasitic nematode species infecting livestock making control increasingly difficult. Wild ruminants are competent hosts of a number of nematode species that typically infect and are best adapted for cattle, sheep, and goats. Thus, the potential exists for wild ruminants to act as reservoirs in the translocation of domestic GIN, including those carrying anthelmintic resistance mutations as well as susceptible genotypes. The potential for parasite exchange is heightened by interfaces or ecotones between managed and wild rangelands, and by perturbations linked to climate warming that can increasingly alter the distributions of wild ungulates and their interactions with domestic and free-ranging ruminants. To investigate the extent to which wild ruminants harbour parasites capable of infecting domestic ruminants we first performed an epidemiological study of feces from wildlife hosts that spanned 16 states and included white-tailed deer (85 % of the samples), pronghorn, elk, mule deer, bighorn sheep, moose, cattle, and caribou across the United States. All samples were cultured to third stage larvae and nematode DNA was isolated and PCR amplified. Among the 548 wild ruminant samples received, 33 % (181 samples) were positive for nematode DNA, among which half (84 samples) contained DNA from GIN species commonly found in cattle. DNA from cattle GIN species was detected in 46 % of samples from the Northeast, 42 % from the Southeast, 10 % from the Midwest, 0 % from the Southwest and 11 % from the West. Deep amplicon sequencing of the ITS-2 rDNA indicated that Ostertagia and Trichostrongylus were present in 90 % and 69 % of the nematode DNA positive samples, respectively, whereas Haemonchus, Cooperia and Oesophagostomum were present in 26 %, 2 % and 10 % of the samples, respectively. These data clearly show that wild ruminants commonly harbour multiple parasite species whose primary hosts are domestic cattle, and suggest that further work is warranted to investigate their specific roles in the management of anthelmintic resistance

A phage-displayed peptide recognizing porcine aminopeptidase N is a potent small molecule inhibitor of PEDV entry

Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluorescence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pre-treatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells. These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells

Molecular taxonomy, phylogeny and biogeography of nematodes belonging to the Trichinella genus

Studying parasites of the genus Trichinella provides scientists of today many advantages. This is a group of zoonotic nematodes that circulates freely among wildlife hosts with one in particular, Trichinella spiralis that is exceptionally well adapted to domestic swine. Recent reports suggest that human infections from hunted animals are on the rise worldwide and numerous countries still experience problems with T. spiralis in their domestic food supplies. Trichinella is a genus whose members are easily propagated in the laboratories, have been used as models to investigate host-parasite relationships and parasitism among clade I organisms, and represent a poorly investigated link between the phylum Nematoda and other Metazoans. The importance of T. spiralis in better understanding the tree of life was so recognized that in 2004, its genome was carefully selected as one of only nine key non-mammalian organisms to be sequenced to completion. Since it was first discovered in 1835, this genus has expanded from being monospecific to eight species including four other genotypes of undetermined taxonomic rank. Inasmuch as discriminating morphological data have been scant, our understanding of the genus has been relegated to a compilation of molecular, biochemical and biological data. Herein, we provide a collection of information and up-to-date interpretations on the taxonomy, diagnostics, systematics, micro- and macroevolution, and the biogeographical and biohistorical reconstruction of the genus Trichinella