Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

Effect of High Pressure Processing on a Wide Variety of Human Noroviruses Naturally Present in Aqua-Cultured Japanese Oysters.

The contamination of oysters with human norovirus (HuNoV) poses a human health risk, as oysters are often consumed raw. In this study, the effect of high pressure processing (HPP) on a wide variety of HuNoVs naturally present in aqua-cultured Japanese oysters was determined through a polymerase chain reaction-based method with enzymatic pretreatment, to distinguish between infectious HuNoV. Among five batches, genogroup I. genotype 1 (GI.1), GI.2, GI.3, and GI.8 HuNoV were detected from only one oyster not treated with HPP in the fifth batch, while genogroup II. genotype 1 to 4 (GII.1 to 4), GII.6, GII.8., GII.9, GII.13, GII.16, GII.17, and GII.22 HuNoV were detected from oysters not treated with HPP in all tested batches as determined by next-generation sequencing analysis. Neither GI nor GII HuNoV was detected in the oysters of any of the batches after HPP treatment. To our knowledge, this is the first study to investigate the effect of HPP on a wide variety of HuNoVs naturally present in aqua-cultured oysters.

Next-Generation Sequencing Analysis of the Diversity of Human Noroviruses in Japanese Oysters.

To obtain detailed information on the diversity of infectious norovirus in oysters (Crossostrea gigas), oysters obtained from fish producers at six different sites (sites A, B, C, D, E, and F) in Japan were analyzed once a month during the period spanning October 2015-February 2016. To avoid false-positive polymerase chain reaction (PCR) results derived from noninfectious virus particles, samples were pretreated with RNase before reverse transcription-PCR (RT-PCR). RT-PCR products were subjected to next-generation sequencing to identify norovirus genotypes in oysters. As a result, all GI genotypes were detected in the investigational period. The detection rate and proportion of norovirus GI genotypes differed depending on the sampling site and month. GII.3, GII.4, GII.13, GII.16, and GII.17 were detected in this study. Both the detection rate and proportion of norovirus GII genotypes differed depending on the sampling site and month. In total, the detection rate and proportion of GII.3 were highest from October to December among all detected genotypes. In January, the detection rates of GII.4 and GII.17 reached the same level as that of GII.3. The proportion of GII.17 was relatively lower from October to December, whereas it was the highest in January. To our knowledge, this is the first investigation on noroviruses in oysters in Japan, based on a method that can distinguish their infectivity.

Effect of High-Pressure Processing on Human Noroviruses in Laboratory-Contaminated Oysters by Bio-Accumulation.

The contamination of oysters with human noroviruses poses a human health risk, since oysters are often consumed raw. In this study, human norovirus genogroup II was allowed to bio-accumulate in oysters, and then the effect of high-pressure processing (HPP) on human noroviruses in oysters was determined through a polymerase chain reaction (PCR)-based method with enzymatic pretreatment to distinguish infectious noroviruses. As a result, oysters could be artificially contaminated to a detectable level of norovirus genome by the reverse transcription-PCR. Concentrations of norovirus genome in laboratory-contaminated oysters were log normally distributed, as determined by the real-time PCR, suggesting that artificial contamination by bio-accumulation was successful. In two independent HPP trials, a 1.87 log10 and 1.99 log10 reduction of norovirus GII.17 genome concentration was observed after HPP at 400 MPa for 5 min at 25°C. These data suggest that HPP is a promising process of inactivation of infectious human noroviruses in oysters. To our knowledge, this is the first report to investigate the effect of HPP on laboratory-contaminated noroviruses in oysters.

Establishment of an equine tetanus antitoxin reference standard for veterinary use in Japan.

To establish the first National Veterinary Assay Laboratory (NVAL) equine tetanus antitoxin reference standard for veterinary use, we manufactured vials of a candidate antitoxin. These were quality tested for moisture content, vacuum, colour, clarity, and the presence of foreign objects. Ultimately, 115 quality-controlled vials were prepared. To estimate the antitoxin potency of the candidate standard, three different laboratories conducted parallel line assays alongside the existing antitoxin standard. These potency estimates ranged from 38 to 42 IU. This activity was maintained for two years after manufacture, as compared with a fresh vial. No statistically significant non-linearity or non-parallelism of the regression lines was observed (p > 0.05). Statistical assessment of inter- and intra-laboratory variability revealed acceptable coefficients of variation of 3.2% and 2.4-3.1%, respectively. Based on these results, the potency of the potential reference standard was calculated at 40 units of antitoxin activity per 1-mL vial. Vials of this preparation were distributed for use as the first equine tetanus antitoxin reference standard for veterinary use in September 2015.

Application of Next-Generation Sequencing to Evaluate the Profile of Noroviruses in Pre- and Post-Depurated Oysters.

The development of procedures for the efficient removal or inactivation of noroviruses from contaminated oysters is of great interest in oyster production. However, there is a critical limitation for evaluating the depuration efficacy of presently available procedures, as no suitable cell culture system currently exists to cultivate noroviruses. Thus, we applied a next-generation sequencing (NGS) technique to characterize norovirus genotypes in pre- and post-depurated oysters. As a result, we revealed the diversity of noroviruses in pre- and post-depurated oysters. Although the applied depuration procedure could reduce the number of bacterial agents to the level recommended by the Japanese Ministry of Health, Labour and Welfare, no significant changes were observed in the detection rate and the proportion of norovirus group (G) I and GII genotypes. To our knowledge, this is the first report to evaluate the profile of noroviruses in pre- and post-depurated oysters, specifically with respect to norovirus removal, using NGS; the findings imply that the removal of noroviruses from oysters through depuration is not presently sufficient. Further studies are needed to develop a more suitable depuration procedure for removing and/or inactivating noroviruses from contaminated oysters.

Application of next-generation sequencing to investigation of norovirus diversity in shellfish collected from two coastal sites in Japan from 2013 to 2014.

A better understanding of the role played by shellfish regarding the manner of pathogen contamination, persistence, and selection may help considering epidemiology of noroviruses. Thus, norovirus genotype profiles in shellfish (Crassostrea gigas and Mitilus galloprovincialis) were investigated by using Next-generation sequencing (NGS) technology. In genogroup I (GI), 7 genotypes (abbreviated as GI.2 to GI.7, and GI.9) were detected from C. gigas, whereas 9 genotypes (GI.1 to GI.9) were detected from M. galloprovincialis. The genotype with the highest proportion found in both C. gigas and M. galloprovincialis was GI.4, and the second highest was GI.3. In genogroup II (GII), 17 genotypes (GII.1 to GII.9, GII.11 to GII.17, GII.21 and GI.22) were detected from C. gigas, whereas 16 genotypes (GII.1 to GII.8, GII.11 to GII.17, GII.21 and GI.22) were detected from M. galloprovincialis. The genotype with the highest proportion in both C. gigas and M. galloprovincialis was GII.4, the next highest differed between C. gigas and M. galloprovincialis. To our knowledge, this study may be the first trial to utilize the latest technology in this field, and reveal the diversity of norovirus genotypes present in shellfish.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish.

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations.

Development of an in vitro assay based on humoral immunity for quality control of oil-adjuvant Pseudotuberculosis vaccine in Yellowtail Seriola quinqueradiata.

Photobacterium damselae subsp. piscicida is an infectious pathogen that causes Pseudotuberculosis in Yellowtail fish. In Japan, several oil-adjuvant vaccines for Pseudotuberculosis have been approved for control of infectious diseases in aquaculture. Before distribution of an approved fish vaccine, an artificial challenge test for quality control is performed by the manufacturer and National Veterinary Assay Laboratory under Pharmaceutical Law of Japan to confirm potency. In this study, artificial challenge tests with a range of five diluted or undiluted approved vaccines was performed to determine the relationship between antigen levels and vaccine efficacy. Immunization of fish with the undiluted vaccine prevented Pseudotuberculosis. Results of artificial challenge tests demonstrated vaccine efficiency was dose dependent. Agglutination assays using immune sera were performed to determine agglutination titers, which were also dose dependent. These results suggest a link between survival rate in the artificial challenge tests and agglutination titers. Western blotting analysis identified a specific protein approximately 37 kDa in size in vaccinated fish. We confirmed antibodies were produced in vaccinated fish by immunoreactions with the approved vaccine. An agglutination assay based on humoral immunoreactions would be a useful alternative to the artificial challenge test for quality control of vaccines for aquaculture.

Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan.

Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD50 values of 0.45-1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan.

Phylogenetic grouping, epidemiological typing, analysis of virulence genes, and antimicrobial susceptibility of Escherichia coli isolated from healthy broilers in Japan.

BACKGROUND: The aim of our study was to investigate the possible etiology of avian colibacillosis by examining Escherichia coli isolates from fecal samples of healthy broilers. FINDINGS: Seventy-eight E. coli isolates from fecal samples of healthy broilers in Japan were subjected to analysis of phylogenetic background, virulence-associated gene profiling, multi-locus sequence typing (MLST), and antimicrobial resistance profiling. Phylogenetic analysis demonstrated that 35 of the 78 isolates belonged to group A, 28 to group B1, one to group B2, and 14 to group D. Virulence-associated genes iutA, iss, cvaC, tsh, iroN, ompT, and hlyF were found in 23 isolates (29.5%), 16 isolates (20.5%), nine isolates (11.5%), five isolates (6.4%), 19 isolates (24.4%), 23 isolates (29.5%), and 22 isolates (28.2%) respectively. Although the genetic diversity of group D isolates was revealed by MLST, the group D isolates harbored iutA (10 isolates, 71.4%), iss (6 isolates, 42.9%), cvaC (5 isolates, 35.7%), tsh (3 isolates, 21.4%), hlyF (9 isolates, 64.3%), iroN (7 isolates, 50.0%), and ompT (9 isolates, 64.3%). CONCLUSIONS: Our results indicated that E. coli isolates inhabiting the intestines of healthy broilers pose a potential risk of causing avian colibacillosis.

Molecular identification and expression analysis of lipocalins from blood feeding taiga tick, Ixodes persulcatus Schulze.

Lipocalins have been known for their several biological activities in blood-sucking arthropods. Recently, the identification and characterization of lipocalins from Ixodes ricinus (LIRs) have been reported and functions of lipocalins are well documented. In this study, we have characterized four Ixodes persulcatus lipocalins that were discovered while analyzing I. persulcatus tick salivary gland EST library. We show that the four I. persulcatus lipocalins, here after named LIPERs (lipocalin from I. persulcatus) are 28.8-94.4% identical to LIRs from I. ricinus. Reverse transcriptase-PCR analysis revealed that lipocalin genes were expressed specifically in the salivary glands throughout life cycle stages of the ticks and were up-regulated by blood feeding. The specific expressions were also confirmed by Western blotting analysis. Furthermore, to investigate whether native lipocalins are secreted into the host during tick feeding, the reactivity of anti-serum raised against saliva of adult ticks to recombinant lipocalins was tested by Western blotting. The lipocalins are potentially secreted into the host during tick feeding as revealed by specific reactivity of recombinant lipocalins with mouse antibodies to I. persulcatus tick saliva. Preliminary vaccination of mice with recombinant lipocalins elicited that period to reach engorgement was significantly delayed and the engorgement weight was significantly reduced as compared to the control. Further elucidation of the biological functions of LIPERs are required to fully understand the pathways involved in the modulation of host immune responses.

Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation.

Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.

Suppression of cell proliferation and cytokine expression by HL-p36, a tick salivary gland-derived protein of Haemaphysalis longicornis.

Previously, a putative immunosuppressant-coding gene was identified from a complementary DNA library derived from the salivary glands of partially-fed Haemaphysalis longicornis. Using real-time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis-derived 36 000 molecular weight protein: rHL-p36). The addition of rHL-p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen-stimulated cells in a dose-dependent manner, with concomitantly significant down-regulation of messenger RNA levels for interleukin-2. The inhibitory response could be abrogated by blockage of HL-p36 with antibody, suggesting the direct involvement of rHL-p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL-p36-inoculated mice was significantly lower than for those from control mice, suggesting that rHL-p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down-regulated by rHL-p36 inoculation. In conclusion, these results suggest that HL-p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.

Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus.

Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK-3 mRNA transcription in comparison to the 1st day. We detected the GSK-3beta isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3beta may be involved in glycogen metabolism regulation during R. microplus embryogenesis.

Quantitative analysis of cytokine mRNA expression and protozoan DNA load in Theileria parva-infected cattle.

Theileria parva (T. parva) causes a highly serious bovine disease called East Coast fever (ECF), which is characterized by pyrexia, dyspnea and cachexia and is of great economic importance in African countries. We hypothesize that the clinical symptoms of ECF could be explained by a cytokine dysregulation. In this study, we investigated the relationship between T. parva DNA load and expression levels of cytokine mRNAs in leukocytes from experimentally infected calves by quantitative PCR. The p104 gene, which encodes the T. parva 104 kDa microneme-rhoptry protein, was detected in cattle blood from day 10 after T. parva-infected tick infestation, and the protozoan DNA load was increased together with severity of disease. The mRNA expressions of pro-inflammatory cytokines, such as interleukin (IL)-1beta and IL-6, were up-regulated with protozoan DNA load increasing. In addition, the level of a type-2 cytokine (IL-10) transcript was also increased during the acute phase. In contrast, the down-regulation or no detectable levels of the expression of type-1 cytokines, such as IL-2 and interferon (IFN)-gamma were observed in T. parva-infected animals. Thus, our observations indicated that high protozoan load and resulting intense inflammatory responses might be involved in the severity of clinical signs observed in T. parva-infection.

Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis.

We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick's defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.

Rhipicephalus appendiculatus: characterization of a testis-associated protein.

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.

Establishment of a laboratory colony of taiga tick Ixodes persulcatus for tick-borne pathogen transmission studies.

Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus-borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20 degrees C with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8 +/- 2 days and had a pre-oviposition period lasting for 7 +/- 2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3 +/- 1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4 +/- 1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus-borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.

Effects of anti-tick cocktail vaccine against Rhipicephalus appendiculatus.

Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36-kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM36 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8% for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8% in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T. parva-infected ticks fed on immunized cattle, the occurrence of T. parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T. parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.