Epidemiology of Severe Fever with Thrombocytopenia Syndrome in Dogs and Cats in Taiwan.

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by the SFTS Virus (SFTSV), is a global health threat. SFTSV in Taiwan has only been reported in ruminants and wild animals. Thus, we aimed to investigate the infection statuses of dogs and cats, the animals with closer human interactions. Overall, the SFTSV RNA prevalence was 23% (170/735), with dogs showing a 25.9% (111/429) prevalence and cats at 19.3% (59/306) prevalence. Noticeably, the prevalence in stray animals (39.8% 77/193) was significantly higher than in domesticated ones (17.2%, 93/542). Among the four categories analyzed, the highest SFTSV prevalence was found in the stray dogs at 53.9% (120/193), significantly higher than the 24.2% prevalence noted in stray cats. In contrast, domesticated animals exhibited similar prevalence rates, with 17.1% for dogs and 17.2% for cats. It is noteworthy that in the domesticated animal groups, a significantly elevated prevalence (45%, 9/20) was observed among cats exhibiting thrombocytopenia compared to those platelet counts in the reference range (4.8%, 1/21). The high infection rate in stray animals, especially stray dogs, indicated that exposure to various outdoor environments influences the prevalence of infections. Given the higher human interaction with dogs and cats, there is a need for proactive measures to reduce the risk associated with the infection of SFTSV in both animals and humans.

In vitro ruminal fermentation and cow-to-mouse fecal transplantations verify the inter-relationship of microbiome and metabolome biomarkers: potential to promote health in dairy cows.

Introduction: There are differences in the gut microbiome and metabolome when the host undergoes different physical or pathological conditions. However, the inter-relationship of microbiome and metabolome biomarkers to potentially promote the health of dairy cows needs to be studied. Further, the development of next-generation probiotics for dairy cattle health promotion has not been demonstrated. Objective: In the present study, we identified the microbiome and metabolome biomarkers associated with healthy cows. Methods: We analyzed the relationships of the ruminal microorganism profile and metabolites between healthy and mastitis lactating dairy cows. The roles of bacterial biomarker were further verified by in vitro fermentation and cow-to-mouse fecal microbiota transplantation (FMT). Results: Two species, Ruminococcus flavefaciens and Bifidobacterium longum subsp. longum, and six rumen metabolites were positively correlated with healthy cows by Spearman's correlation analysis. Through in vitro ruminal fermentation, inoculating R. flavefaciens and B. longum subsp. longum showed the upregulation of the levels of putrescine, xanthurenic acid, and pyridoxal in the mastitis ruminal fluid, which confirmed the inter-relationships between these microbiota and metabolites associated with healthy cows. Further, we verified the role of R. flavefaciens and B. longum subsp. longum in promoting health by FMT. The administration of R. flavefaciens and B. longum subsp. longum reduced the death rate and recovered the bodyweight loss of germ-free mice caused by FMT mastitis feces. Discussion: We provided evidence that the bacterial biomarkers alter downstream metabolites. This could indirectly indicate that the two bacterial biomarkers have the potential to be used as next-generation probiotics for dairy cattle, although it needs more evidence to support our hypothesis. Two species, R. flavefaciens and B. longum subsp. longum, with three metabolites, putrescine, xanthurenic acid, and pyridoxal, identified in the ruminal fluid, may point to a new health-promoting and disease-preventing approach for dairy cattle.

The First Nationwide Surveillance of Severe Fever with Thrombocytopenia Syndrome in Ruminants and Wildlife in Taiwan.

Since the first discovery of severe fever with thrombocytopenia syndrome virus (SFTSV) in China in 2009, SFTSV has rapidly spread through other Asian countries, including Japan, Korea, Vietnam and Pakistan, in chronological order. Taiwan reported its first discovery of SFTSV in sheep and humans in 2020. However, the prevalence of SFTSV in domestic and wildlife animals and the geographic distribution of the virus within the island remain unknown. A total of 1324 animal samples, including 803 domestic ruminants, 521 wildlife animals and 47 tick pools, were collected from March 2021 to December 2022 from 12 counties and one terrestrial island. The viral RNA was detected by a one-step real-time reverse transcription polymerase chain reaction (RT-PCR). Overall, 29.9% (240/803) of ruminants showed positive SFTSV RNA. Sheep had the highest viral RNA prevalence of 60% (30/50), followed by beef cattle at 28.4% (44/155), goats at 28.3% (47/166), and dairy cows at 27.5% (119/432). The bovine as a total of dairy cow and beef cattle was 27.8% (163/587). The viral RNA prevalence in ticks (predominantly Rhipicephalus microplus) was similar to those of ruminants at 27.7% (13/47), but wild animals exhibited a much lower prevalence at 1.3% (7/521). Geographically the distribution of positivity was quite even, being 33%, 29.1%, 27.5% and 37.5% for northern, central, southern and eastern Taiwan, respectively. Statistically, the positive rate of beef cattle in the central region (55.6%) and dairy cattle in the eastern region (40.6%) were significantly higher than the other regions; and the prevalence in Autumn (September-November) was significantly higher than in the other seasons (p < 0.001). The nationwide study herein revealed for the first time the wide distribution and high prevalence of SFTSV in both domestic animals and ticks in Taiwan. Considering the high mortality rate in humans, surveillance of other animal species, particularly those in close contact with humans, and instigation of protective measures for farmers, veterinarians, and especially older populations visiting or living near farms or rural areas should be prioritized.

Deletion of gene OV132 attenuates Orf virus more effectively than gene OV112.

Orf virus (ORFV), a Parapoxvirus in Poxviridae, infects sheep and goats resulting in contagious pustular dermatitis. ORFV is regarded as a promising viral vector candidate for vaccine development and oncolytic virotherapy. Owing to their potential clinical application, safety concerns have become increasingly important. Deletion of either the OV132 (encoding vascular endothelial growth factor, VEGF) or OV112 (encoding the chemokine binding protein, CBP) genes reduced ORFV infectivity, which has been independently demonstrated in the NZ2 and NZ7 strains, respectively. This study revealed that the VEGF and CBP gene sequences of the local strain (TW/Hoping) shared a similarity of 47.01% with NZ2 and 90.56% with NZ7. Due to the high sequence divergence of these two immunoregulatory genes among orf viral strains, their contribution to the pathogenicity of Taiwanese ORFV isolates was comparatively characterized. Initially, two ORFV recombinants were generated, in which either the VEGF or CBP gene was deleted and replaced with the reporter gene EGFP. In vitro assays indicated that both the VEGF-deletion mutant ORFV-VEGFΔ-EGFP and the CBP deletion mutant ORFV-CBPΔ-EGFP were attenuated in cells. In particular, ORFV-VEGFΔ-EGFP significantly reduced plaque size and virus yield compared to ORFV-CBPΔ-EGFP and the wild-type control. Similarly, in vivo analysis revealed no virus yield in the goat skin biopsy infected by ORFV-VEGFΔ-EGFP, and significantly reduced the virus yield of ORFV-CBPΔ-EGFP relative to the wild-type control. These results confirmed the loss of virulence of both deletion mutants in the Hoping strain, whereas the VEGF-deletion mutant was more attenuated than the CBP deletion strain in both cell and goat models. KEY POINTS: • VEGF and CBP genes are crucial in ORFV pathogenesis in the TW/Hoping strain • The VEGF-deletion mutant virus was severely attenuated in both cell culture and animal models • Deletion mutant viruses are advantageous vectors for the development of vaccines and therapeutic regimens.

Prevalence, Virulence, and Antimicrobial Resistance of Major Mastitis Pathogens Isolated from Taiwanese Dairy Farms.

Mastitis, a highly prevalent disease in dairy cows, is responsible for massive financial losses due to decreased milk yield, milk quality, and costly medication. This research paper investigates antimicrobial susceptibility in cows and the role played by both resistance and virulence gene distribution in bovine mastitis. A total of 984 raw milk samples were collected from five different dairy farms and cultured on sheep blood agar plates. Antimicrobial susceptibility was determined by disc diffusion, and corresponding resistance and virulence genes were detected by PCR. Among the collected milk samples, 73, 32, and 19 isolates of Streptococcus spp., Staphylococcus spp., and coliforms were identified, respectively. The antimicrobial susceptibility results showed that Streptococcus spp. were resistant to tetracycline (86.30%), neomycin (79.45%), and oxacillin (73.97%). Staphylococcus spp. were resistant to tetracycline (59.37%) and oxacillin (53.12%). Lastly, coliforms were resistant to oxacillin (100%) and bacitracin (68.42%). The genotyping results showed that Streptococcus spp. carried the resistance genes tetM (46.57%) against tetracycline, bcrB (41.09%) against bacitracin, and aph(3)-II (39.72%) against neomycin. Staphylococcus spp. carried the resistance genes bcrB (40.62%) and tetM (18.75%), and coliforms carried the resistance genes tetM (42.10%) and bcrB (57.89%). Moreover, 57.53%, 75.0%, and 63.15% of Streptococcus spp., Staphylococcus spp., and coliforms carried lmb, fib, and ompC virulence genes, respectively. All three tested bacterial genera showed no significant association between antimicrobial resistance genes and virulence factors, although they were negatively correlated (p > 0.05). The combination of resistance gene identification and susceptibility tests as components of the diagnosis of bovine mastitis can help in selecting effective antimicrobial agents to treat it.

Prototheca Infections and Ecology from a One Health Perspective.

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.

Development of Next-Generation Probiotics by Investigating the Interrelationships between Gastrointestinal Microbiota and Diarrhea in Preruminant Holstein Calves.

(1) Background: We aimed to isolate and identify potential next-generation probiotics (NGP) by investigating the interrelationships between gastrointestinal microbiota and diarrhea in preruminant Holstein calves. (2) Material and methods: Twenty preruminant Holstein calves were divided into healthy and diarrheic groups after the combination outcomes of veterinary diagnosis and fecal scores. The fecal microbiome, plasma cytokines, plasma immunoglobulin (Ig) G and haptoglobin were analyzed. The potential probiotic bacteria were identified by comparing the microbiota difference between healthy and diarrheic calves and correlation analysis with fecal scores and inflammatory markers. The identified bacteria were also isolated for further evaluation for antimicrobial activities and immunoregulatory effects. (3) Results: Microbiota analysis suggested that Ruminococcaceae_UCG_014, Bifidobacterium and Pseudoflavonifractor positively correlated with bovine IgG and negatively correlated with fecal score; inflammatory factors, bovine HP, and IL-8 were classified as beneficial bacteria contributing to the health of the calves. The alternation of gut microbial composition also induced changes in the functional gene enrichment of gut microbiota in calves. The gathering of microbiomic data strongly indicated the possible beneficial effects of Bifidobacterium longum subsp. longum, expected to develop as NGP. After isolation and evaluation of the potential functionality in vitro, two specific bifidobacterial strains demonstrated antimicrobial activities and immunoregulatory effects. (4) Conclusions: The results provide a new probiotic searching approach for preventing gastrointestinal disorders in preruminant calves. Further animal study is necessary to verify the results.

Potential Risk Factors Associated with Infection with Bovine Leukaemia Virus in Dairy and Beef Cattle in Taiwan.

Bovine leukaemia virus (BLV), which is classified as a Deltaretrovirus, is the aetiologic agent of enzootic bovine leukosis (EBL), a chronic lymphoproliferative disorder with a worldwide distribution. EBL is widespread in dairy herds and causes a direct economic impact due to reduced milk production and the early culling of BLV-infected cattle. The BLV infection status in Taiwan remains largely unknown; a high prevalence of BLV in dairy cows was recently revealed. The present study further investigated BLV infections in beef cattle. Surprisingly, the prevalence of BLV proviral DNA was as low as 11.8% (23/195), which is significantly lower than that noted in dairy cows, which was 42.5% (102/240) (p < 0.001). Factors associated with BLV infections were subsequently investigated. Due to the differences in herd management, an analysis of risk factors for a BLV infection was independently conducted in these two sectors. Several factors associated with a BLV infection were identified. Age was significantly associated with BLV infection status in dairy cows (p < 0.001) but not in beef cattle. A high prevalence of BLV was observed in cattle >15.5 months old (57.8%) compared with those ≤15.5 months old (11.4%). Moreover, after stratification analysis, based on the critical age of 15.5 months, as determined by the receiver operating characteristic (ROC) curve, a significantly higher BLV prevalence was demonstrated in lactating dairy cows, cattle undergoing bull breeding, heifers at older ages, and those undergoing routine rectal palpation. Due to the high prevalence of BLV in Taiwan, the development of an effective control program, based on the identified risk factors, is important for interrupting the routes of BLV transmission within herds.

Investigation of Transovarial Transmission of Bartonella henselae in Rhipicephalus sanguineus sensu lato Ticks Using Artificial Feeding.

Bartonella henselae is a slow-growing, Gram-negative bacterium that causes cat scratch disease in humans. A transstadial transmission of the bacteria from larvae to nymphs of Rhipicephalus sanguineus sensu lato (s.l.) ticks, suspected to be a potential vector of the bacteria, has been previously demonstrated. The present study aims to investigate transovarial transmission of B. henselae from R. sanguineus s.l. adults to their instars. Adult ticks (25 males and 25 females) were fed through an artificial feeding system on B. henselae-infected goat blood for 14 days, and 300 larvae derived from the experimentally B. henselae-infected females were fed on noninfected goat blood for 7 days. Nested PCR and culture were used to detect and isolate B. henselae in ticks and blood samples. Bartonella henselae DNA was detected in midguts, salivary glands, and carcasses of the semi-engorged adults and pooled tick feces (during feeding and post-feeding periods). After the oviposition period, B. henselae DNA was detected in salivary glands of females (33.3%), but not in pooled eggs or larvae derived from the infected females. However, B. henselae DNA was detected by nested PCR from the blood sample during larval feeding, while no viable B. henselae was isolated by culture. According to our findings, following infected blood meal, B. henselae could remain in the tick midguts, move to other tissues including salivary glands, and then be shed through tick feces with limited persistency. The presence of bacterial DNA in the blood during larval feeding shows the possibility of transovarial transmission of B. henselae in R. sanguineus s.l. ticks.

Investigating the Reciprocal Interrelationships among the Ruminal Microbiota, Metabolome, and Mastitis in Early Lactating Holstein Dairy Cows.

Mastitis in dairy cow significantly affects animal performance, ultimately reducing profitability. The reciprocal interrelationships among ruminal microbiota, metabolome, and mastitis combining early inflammatory factors (serum proinflammatory cytokines) in lactating dairy cows has not been explored, thus, this study evaluated these reciprocal interrelationships in early lactating Holstein dairy cows to identify potential microbial biomarkers and their relationship with ruminal metabolites. The ruminal fluid was sampled from 8 healthy and 8 mastitis cows for the microbiota and metabolite analyses. The critical ruminal microbial biomarkers and metabolites related to somatic cell counts (SCC) and serum proinflammatory cytokines were identified by the linear discriminant analysis effect size (LEfSe) algorithm and Spearman's correlation analysis, respectively. The SCC level and proinflammatory cytokines positively correlated with Sharpea and negatively correlated with Ruminococcaceae UCG-014, Ruminococcus flavefaciens, and Treponema saccharophilum. Furthermore, the metabolites xanthurenic acid, and 1-(1H-benzo[d]imidazol-2-yl) ethan-1-ol positively correlated with microbial biomarkers of healthy cows, whereas, xanthine, pantothenic acid, and anacardic acid were negatively correlated with the microbial biomarkers of mastitis cows. In conclusion, Ruminococcus flavefaciens and Treponema saccharophilum are potential strains for improving the health of dairy cows. The current study provides a novel perspective to assist in targeting the ruminal microbiota with preventive/therapeutic strategies against inflammatory diseases in the future.

Prototheca from bovine milk and associated minimal algaecide concentration of chlorhexidine and povidone-iodine in Taiwan.

OBJECTIVE: Protothecal mastitis has emerged and affected the dairy industries globally in last decades; however, little is known about the occurrence of Prototheca in Taiwan. The present study identified the Prototheca isolated from bovine milk and revealed the algaecide efficacy of 2 disinfectants. MATERIAL AND METHODS: From 2011 to 2014, a total of 176 Prototheca isolates were obtained from 10 625 milk samples collected from dairy cows in 24 Taiwanese dairy farms. Of these, 149 isolates were identified by biochemical tests and genotype differentiation with a specific PCR protocol. Minimal algaecide concentration (MAC) of chlorhexidine and povidone-iodine were obtained from 16 isolates by microdilution method. RESULTS: Almost all (n = 147) isolates were P. zopfii, and remaining 2 isolates were P. wickerhamii and unidentified, respectively. Based on molecular genotyping, all P. zopfii isolates belonged to genotype 2. The MAC of 2 commonly used disinfectants, chlorhexidine and povidone-iodine, against 16 P. zopfii isolates was 1.56-3.13 and 48.83-390.63 µg/ml, respectively. CONCLUSION: This report revealed for the first time the prevalence and the predominant P. zopfii genotype 2 of bovine protothecal infection in Taiwan. In vitro algaecide efficacy indicated that both tested disinfectants were effective against P. zopfii.

The Rumen Specific Bacteriome in Dry Dairy Cows and Its Possible Relationship with Phenotypes.

Most microbiome studies of dairy cows have investigated the compositions and functions of rumen microbial communities in lactating dairy cows. The importance of the relationships among hosts, microbiota, diet composition, and milk production remains unknown in dry dairy cows. Thus, in the present study, the composition of the rumen microbiome in cows from three dairy farms was investigated to identify core bacteria contributing to various physiological roles during rumen fermentation in dry dairy cows. The results indicated that ruminal fluid in dry dairy cows from different regional farms had core rumen microbiota that could be clearly distinguished from that of cows of the other farms. Further identification of key microorganisms associated with each farm revealed that Prevotella, Methanobrevibacter, Pseudobutyrivibrio, Ruminococcus, Bacteroides, and Streptococcus were major contributors. Spearman's correlation indicated that the abundance of genera such as Prevotella and Ruminococcus in dry dairy cows could indicate milk yield in the previous lactating period. Functional pathway analysis of the rumen bacterial communities demonstrated that amino acid metabolism and carbohydrate metabolism were the major pathways. Our findings provide knowledge of the composition and predicted functions of rumen microbiota in dry dairy cows from regional farms, which underscore the importance of the relationships among hosts, microbiota, diet composition, and milk production.

Transmission of Bartonella henselae within Rhipicephalus sanguineus: Data on the Potential Vector Role of the Tick.

Bartonella henselae is a fastidious intraerythrocytic, gram-negative bacteria that causes cat scratch disease in humans. Ixodes ricinus has been confirmed to be a competent vector of B. henselae, and some indirect evidences from clinical cases and epidemiological studies also suggested that some other tick species, including Rhipicephalus sanguineus, may transmit the bacteria. B. henselae has been detected in R. sanguineus but no experimental investigations have been performed to evaluate the vector competency of this tick species regarding B. henselae transmission. To this end, this work aimed to assess the transstadial transmission of B. henselae between larvae and nymphs of R. sanguineus as well as transmission by nymphs infected at the larval stage. Four hundred B. henselae negative larvae were fed with B. henselae-infected blood by using an artificial membrane feeding system. After five days of feeding, B. henselae was detected by PCR in 57.1% (8/14) of engorged larval pools, 66.7% (4/6) of semi-engorged larval pools, and 66.7% (2/3) of larval feces pools. After molting, B. henselae DNA was also detected in 10% (1/10) of nymph pools, but not in tick feces. After a pre-fed step of nymphs infected at the larval stage on non-infected blood meal, B. henselae was detected by PCR in blood sample from the feeder, but no Bartonella colonies could be obtained from culture. These findings showed that B. henselae could be transstadial transmitted from R. sanguineus larvae to nymphs, and also suggest that these nymphs may retransmitted the bacteria through the saliva during their blood meal. This is the first study that validated the artificial membrane feeding system for maintaining R. sanguineus tick colony. It shows the possibility of transstadial transmission of B. henselae from R. sanguineus larvae to nymphs.

Molecular Epidemiological and Serological Studies of Bovine Leukemia Virus in Taiwan Dairy Cattle.

Bovine leukemia virus (BLV) infection results in a decrease in milk yield and quality, a compromise in immunity, and shortening in the longevity of cows. The current status of BLV infection of dairy cattle in Taiwan remains unclear. To evaluate BLV infection, anti-BLV gp51 antibody and proviral DNA were detected. Surprisingly, the seroprevalence of BLV at the animal and herd level was as high as 81.8% (540/660 cattle) and 99.1% (109/110 herds), respectively. Among 152 blood samples analyzed, 132 (86.8%) were detected as positive for BLV-proviral DNA. When the complete blood count (CBC) was taken into account, the white blood cell (WBC) number appears to be the factor with the highest predicted potential for BLV infection. Moreover, based on receiver operating characteristic (ROC) curve analysis, the sensitivity and specificity are 72.0 and 75.0%, respectively, when the cut-off value of the WBC was set at 10.215 K/µL. Despite the co-circulation of genotype 1 and 3 in Taiwan, genotype 1 was much more prevalent (29/30). Taken together, due to the high prevalence of BLV, the identification of risk factors for interrupting the routes of transmission of BLV are critical for the control and prevention of further BLV infection.

Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great utility in diagnostic and surveillance activities in which test accuracy is of utmost importance for effective disease intervention.

Virus-like particle secretion and genotype-dependent immunogenicity of dengue virus serotype 2 DNA vaccine.

UNLABELLED: Dengue virus (DENV), composed of four distinct serotypes, is the most important and rapidly emerging arthropod-borne pathogen and imposes substantial economic and public health burdens. We constructed candidate vaccines containing the DNA of five of the genotypes of dengue virus serotype 2 (DENV-2) and evaluated the immunogenicity, the neutralizing (Nt) activity of the elicited antibodies, and the protective efficacy elicited in mice immunized with the vaccine candidates. We observed a significant correlation between the level of in vitro virus-like particle secretion, the elicited antibody response, and the protective efficacy of the vaccines containing the DNA of the different DENV genotypes in immunized mice. However, higher total IgG antibody levels did not always translate into higher Nt antibodies against homologous and heterologous viruses. We also found that, in contrast to previous reports, more than 50% of total IgG targeted ectodomain III (EDIII) of the E protein, and a substantial fraction of this population was interdomain highly neutralizing flavivirus subgroup-cross-reactive antibodies, such as monoclonal antibody 1B7-5. In addition, the lack of a critical epitope(s) in the Sylvatic genotype virus recognized by interdomain antibodies could be the major cause of the poor protection of mice vaccinated with the Asian 1 genotype vaccine (pVD2-Asian 1) from lethal challenge with virus of the Sylvatic genotype. In conclusion, although the pVD2-Asian 1 vaccine was immunogenic, elicited sufficient titers of Nt antibodies against all DENV-2 genotypes, and provided 100% protection against challenge with virus of the homologous Asian 1 genotype and virus of the heterologous Cosmopolitan genotype, it is critical to monitor the potential emergence of Sylvatic genotype viruses, since vaccine candidates under development may not protect vaccinated humans from these viruses. IMPORTANCE: Five genotype-specific dengue virus serotype 2 (DENV-2) DNA vaccine candidates were evaluated for their immunogenicity, homologous and heterologous neutralizing (Nt) antibody titers, and cross-genotype protection in a murine model. The immunity elicited by our prototype vaccine candidate (Asian 1 genotype strain 16681) in mice was protective against viruses of other genotypes but not against virus of the Sylvatic genotype, whose emergence and potential risk after introduction into the human population have previously been demonstrated. The underlying mechanism of a lack of protection elicited by the prototype vaccine may at least be contributed by the absence of a flavivirus subgroup-cross-reactive, highly neutralizing monoclonal antibody 1B7-5-like epitope in DENV-2 of the Sylvatic genotype. The DENV DNA vaccine directs the synthesis and assembly of virus-like particles (VLPs) and induces immune responses similar to those elicited by live-attenuated vaccines, and its flexibility permits the fast deployment of vaccine to combat emerging viruses, such as Sylvatic genotype viruses. The enhanced VLP secretion obtained by replacement of ectodomain I-II (EDI-II) of the Cosmopolitan genotype vaccine construct (VD2-Cosmopolitan) with the Asian 1 EDI-II elicited significantly higher total IgG and Nt antibody titers and suggests a novel approach to enhance the immunogenicity of the DNA vaccine. A DENV vaccine capable of eliciting protective immunity against viruses of existing and emerging genotypes should be the focus of future DENV vaccine development.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, ß, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Strategy for the treatment of puerperal metritis and improvement of reproductive efficiency in cows with retained placenta.

The objective of this study was to improve the reproductive efficiency of dairy cows with puerperal metritis (PM) subsequent to retained placenta (RP) using a two-step treatment strategy. A total of 188 postpartum cows, aged from 2 to 8 years, were utilised for 2 experiments. In Experiment 1, cows affected with RP/PM were randomly assigned to two treatment groups. Cows in Group A (n = 17) were treated with 600 mg of ceftiofur intramuscularly for 3 days followed by intrauterine lavage with 0.1% chlorhexidine and infusion with 0.5% povidoneiodine, while cows in Group B (n = 16) received two intrauterine infusions, first with 5 g of oxytetracycline and then with 0.5% povidone-iodine. Cows with normal postpartum findings were regarded as the healthy control group (n = 26). Ultrasonographic examination revealed that the ovarian activities including the appearance of a dominant follicle and days to first ovulation of the cows in Group A during the early postpartum period differed from those of Group B (P < 0.05), which coincided with the results of uterine swabbing for bacteriology. In Experiment 2, cows with normal postpartum findings were allocated to Group D (n = 78), which received an ovulation protocol (GnRH - 7 d PGF2α - 48 h hCG - 24 h AI) on day 50 ± 2 postpartum. Cows affected with PM were randomly divided into two groups, Group E (n = 25) combined the treatments applied in Groups A and D, while Group F (n = 26) repeated the treatment administered in Group E except for uterine lavage. The results indicated that the pregnancy rate within 150 days postpartum and the mean days open in Group E (76.0% and 106.3 ± 4.6 days, respectively) were significantly different from those in Group F (38.5% and 137.9 ± 10.9 days, respectively) (P < 0.05). This study suggests that reproductive efficiency could be improved by using the two-step treatment to regulate uterine involution and an early resumption of ovarian function in cows with RP/PM.

Bartonella species and their ectoparasites: selective host adaptation or strain selection between the vector and the mammalian host?

A wide range of blood-sucking arthropods have either been confirmed or are suspected as important vectors in Bartonella transmission to mammals, including humans. Overall, it appears that the diversity of Bartonella species DNA identified in ectoparasites is much broader than the species detected in their mammalian hosts, suggesting a mechanism of adaptation of Bartonella species to their host-vector ecosystem. However, these mechanisms leading to the fitness between the vectors and their hosts still need to be investigated.