P. aeruginosa (PAO1) and S. aureus (SAP231) biofilms in in vitro assays. Antibiotic loaded β-TCP/CS beads were compared with antibiotic loaded beads formed from a well characterized synthetic calcium sulfate (CS) bone void filler. β-TCP/CS antibiotic loaded showed antimicrobial potency against PAO1 in a repeated Kirby-Bauer like zone of inhibition assay for 6 days compared to 8 days for CS. However, both bead types showed potency against SAP231 for 40 days. Both formulations loaded with V + T completely prevented biofilm formation (CFU below detection limits) for the 3 days of the experiment with daily fresh inoculum challenges (P < 0.001). In addition, both antibiotic loaded materials and antibiotic combinations significantly reduced the bioburden of pre-grown biofilms by between 3 and 5 logs (P < 0.001) with V + G performing slightly better against PAO1 than V + T. Our data, combined with previous data on osteogenesis suggest that antibiotic loaded β-TCP/CS may have potential to stimulate osteogenesis through acting as a scaffold as well as simultaneously protecting against biofilm infection. Future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate the use of β-TCP/CS in the management of orthopaedic infections.This study investigated the efficacy of a biphasic synthetic β-tricalcium phosphate/calcium sulfate (β-TCP/CS) bone graft substitute for compatibility with vancomycin (V) in combination with tobramycin (T) or gentamicin (G) evidenced by the duration of potency and the prevention and killing efficacies of  As for FRI, it’s estimated that the FRI incidence ranges anywhere from 0.4 to 16.1%, with an average of 5%2. Despite many efforts, current clinical efficacy of PJI and FRI, especially regarding the incidence of infection relapse4, remains unsatisfactory, which is primarily attributed to the presence of difficult-to-eliminate biofilms6. Therefore, how to effectively treat the biofilms is the key to lower the risk of reinfections and improve treatment efficacy8.Orthopaedic implant associated infections, including periprosthetic joint infections (PJI) and fracture-related infections (FRI), still pose great challenges to orthopaedic surgeons. According to a recent study9 has shown improved efficacy following combinations of antibiotic-loaded CS and PL compared with these interventions alone, in removing P. aeruginosa and S. aureus biofilms attached to the surfaces of stainless steel. Although the primary function of ALBC is structural integrity in fixation or as a spacer, it can also be used in the form of beads with the primary function of local antibiotic release10. Compared with PMMA, CS beads has many advantages, such as carrying a wider range antibiotics such as those which may be heat sensitive, being completely biodegradable and not requiring a second surgery for removal11. Therefore, antibiotic-loaded CS (ALCS) has been used in the management of implant associated infections13.Clinically, one of the most frequently used management strategies in orthopaedic device associated infections is radical debridement and wound pulse lavage (PL), followed by local implantation of antibiotic-loaded poly methylmethacrylate (PMMA) bone cement (ALBC) or absorbable mineral based materials such as calcium sulfate (CS). Previous work from our lab17 and FRI21 following joint arthroplasty and open fracture surgery, respectively. After analyzing the prophylactic effect of ALBC against infection in primary cemented TKA of 731,214 cases, Jameson et al.16 concluded that ALBC was associated with a lower risk of revisions for infections . As for FRI, a recent systematic review22 summarized the effect of local antibiotic prophylaxis when treating open limb fractures. Outcomes also showed a decreased FRI risk of 12% when additional local antibiotics were provided prophylactically. Whilst most of these studies focused on PMMA, clinical evidence of absorbable based mineral materials in primary prophylaxis of bone and joint surgery remains limited.In addition to infection management, absorbable based mineral materials or ALBC can also provide early protection against biofilm formation after primary osteoarticular surgery. Previous studies have indicated that local use of ALBC is effective in preventing both PJI23. In previous in vitro studies, we have demonstrated synthetic pharmaceutical grade CS is compatible with a wide range of antibiotics used alone or in dual combinations23 and that the release of antibiotics at potent levels can be sustained over multiple weeks and can significantly prevent or retard the establishment of biofilms and eradicate or significantly reduce the numbers of bacteria in pre-established biofilms of Gram-negative and Gram-positive pathogens common to PJI26. Here we assessed the compatibility and potency of a novel biphasic absorbable material composed of 50% beta-tricalcium phosphate and 50% CS (β-TCP/CS) for controlling biofilms. This biphasic β-TCP/CS material has been designed to stimulate osteogenic activity and accelerate bone formation28. Here we assessed the antimicrobial potency when β-TCP/CS in the form of beads were loaded with vancomycin (V) in combination with tobramycin (T) or gentamicin (G). We compared antibiotic-loaded β-TCP/CS beads with the well characterized antibiotic-loaded CS beads in potency duration and the prevention and killing efficacy of P. aeruginosa and S. aureus biofilms.Absorbable mineral-based beads are primarily used as bone void fillers to manage dead space and promote bone growth. In a similar manner in which orthopaedic surgeons have mixed antibiotics into bone cement to add antimicrobial functionality to PMMA, antibiotics can also be added to mineral bone void fillersP. aeruginosa wild type PAO129 and S. aureus SAP231 (a bioluminescent transformed USA 300 strain)30 were used in this study. PAO1 and SAP231 were grown in Tryptic Soy Broth  and Brain Heart Infusion broth , respectively, at 37˚C overnight on shaker conditions set at 200 rpm. The minimum inhibitory concentration (MIC) of vancomycin (V), tobramycin (T) and gentamicin (G) was determined by microbroth dilution31 using a 96 well-plate and two-fold dilutions from 0.125 to 64 for T and G and up to 1024 for V. OD600 was measured by plate reader and the concentration at which the OD was < 0.1 used as the breakpoint MIC value. The MIC of V,T and G against SAP231 was 4.0, 32.0 and 32.0 µg/mL respectively. The MIC of T and G against PAO1 were 4.0 μg/mL, but this strain was highly resistant to vancomycin (V) with a MIC > 1024 μg/mL.32. All antibiotics were obtained from GoldBio . The procedures for preparation of the beads have been described previously9. The beads were 4.8 mm in diameter approximately 0.108 g in weight, and each antibiotic-loaded bead would be expected to contain on average 4.13 mg of vancomycin and 1.02 mg of tobramycin or gentamicin.Antibiotic-loaded Beads (ALB) were prepared using Genex and Stimulan Rapid Cure products . Genex is a synthetic biphasic material composed of β-TCP and CS in a weight ratio of 1:1, and Stimulan is a high purity CS product. The mixture ratio for V + T and V + G groups were 1000 mg vancomycin and 240 mg tobramycin, and 1000 mg vancomycin and 240 mg gentamicin, per 10 cc (~ 16 g) Genex or Stimulan (~ 20 g) powder, respectively, for clinical relevance24, was used to determine the potency of ALB over time. Overnight cultures of PAO1 and SAP231 were diluted to 1% in TSB and BHI broth, respectively. 100 µL of diluted culture was then spread on either Tryptic Soy Agar (TSA) or BHI agar plates, followed by centrally placing ALB with either V + T or V + G on top of the agar with sterile forceps. The plates were incubated at 37˚C and 5% CO2 for 24 h and zones of inhibition (ZOI) were subsequently analyzed and recorded. Subsequently, the beads were then transferred to a newly prepared bacterial lawn. The procedure was repeated each day until the ZOI was zero. The maximum diameter of the ZOI was measured every 24 h with a ruler, and then converted into units of area (cm2) using the formula A = πr2. Where A is the area of the circle, r is the radius and π is a constant . Area was used rather than diameter since heterogeneity in the beads can sometimes lead to irregular shaped ZOI.A revised Kirby-Bauer test, as described previously6 CFU/mL and incubated on a shaker set at 37 °C and 50 rpm. Every 24 h, the spent media were replaced, and the beads were subjected to a fresh bacterial challenge of 2 mL of 106 CFU/mL.ALB and unloaded beads (10 beads per well) were placed into 6-well plates  and 35 mm MatTek tissue culture plates . We used the 35 mm MatTek plates specifically for those biofilms in the prevention assay to be imaged on the confocal microscope to be compatible with our stage holder. Each well was inoculated with 4 mL of an overnight culture diluted with fresh TSB for PAO1 or BHI for SAP231 to achieve a concentration of approximately 106 CFU/mL of PAO1 or 4 mL BHI and ~ 106 CFU/mL SAP231. Biofilms were allowed to establish for 3 days (37 °C and 50 rpm) with daily media exchanges of 2 mL fresh media. After 72 h, 10 ALB or unloaded beads per well were added to each plate along with fresh media. Every 24 h, the spent media was replaced. The effect of the beads on the amount of biofilm was assessed by viable count using the plate count method.Initially, each well of a 6-well plate was inoculated with either 4 mL of TSB and ~ 1033. Wells with bacteria-inoculated media without any beads  or unloaded beads  were used as negative controls.Individual wells were rinsed twice with 4 mL phosphate buffered saline (PBS)  to remove planktonic cells. The surface of each well was scraped using a cell scraper  to remove the attached biofilms into 1 mL PBS. Samples were vortexed for 20 s to homogenize the biofilm bacteria, tenfold serial dilutions were performed in PBS and plated onto TSA (for PAO1) and BHI (for SAP231) agar plates using the drop plate method, in triplicateTo support the CFU data from the prevention assay, CLSM  was performed on biofilms growing in the MatTek plates. At days 1 and 3, using the fluorescent stain SYTO9 , which stains both live and dead cells green to microscopically quantify and statistically compare the biofilm surface area. Each plate was rinsed with PBS (as detailed in the viable cell count) and stained with 1 µl of SYTO9 per 1 mL of PBS for 20 min, according to manufacturer’s instructions. Then, the plates were gently rinsed with PBS once more and finally 1 mL of PBS was added to the well before observation. In each group, three CLSM images were selected and ImageJ (NIH) software was used to measure the surface areas of the biofilms.10 transformed and the student t test or one-way analysis of variance (ANOVA) was firstly used to evaluate the effect of single independent factor on the CFU count. F-test was used in case of homoscedasticity, and Bonferroni method was used for subsequent post-hoc multiple comparisons. While Welch test and Dunnett’s T3 method was applied, respectively in case of heteroscedasticity. Then, multivariate analysis of variance (MANOVA) was used to investigate the influences of treatment, exposure time, bacteria strain and antibiotic carrier on CFU count as well as potential interactions among the four independent factors, sorted by the prevention and killing groups, respectively. Differences were considered statistically significance when P ≤ 0.05.Statistical analysis was conducted using the Statistical Product and Service Solutions (SPSS) software (version 19.0) . The CFU data was LogPAO1 was susceptible to both V + T and V + G released from CS and β-TCP/CS beads, producing a large ZOI after 1 day  with between approximately 8 and 9-log reductions for days 1 and 3 with both antibiotic combinations compared to the controls  . However, the antibiotic carrier showed no significant effect (P = 0.558), but the other three factors  revealed significant contributions to the CFU count. In addition to their independent effect on the CFU count, all of the four factors  demonstrated a significant interaction effect on the CFU count  Figs. . The oveP < 0.001) with similar reductions observed after 72 h   , but only the treatment strategy and antibiotic carrier contributed significantly to the CFU count (P < 0.001) individually. In addition, significant interactions were found between treatment with exposure time, treatment with bacteria strain, treatment with antibiotic carrier, and exposure time with antibiotic carrier. Moreover, such significant interactions were also observed among treatment, exposure time and antibiotic carrier, as well as among all of the four independent factors  to assess how this formulation compared to that of synthetic CS. We combined two frequently used antibiotic combinations used in bone cements or mineral based bone void fillers, vancomycin, tobramycin and gentamicin34, and we also selected common representatives of Gram positive and negative isolated bacteria from clinical infection cases, S. aureus and P. aeruginosa35 as challenge pathogens to assess efficacy of antibiotic loaded β-tricalcium phosphate/calcium sulfate (β-TCP/CS) beads. The performance of V + T by CS beads against PAO1 was similar to our previous study25 in terms of the duration and potency of the beads which showed efficacy for 8 days. Similarly, the ZOI profile with SAP231 was in accordance with previously published data25. Comparing the results of β-TCP/CS to those of CS beads in the current study suggests that β-TCP/CS beads possesses similar levels and durations of antimicrobial activity against two pathogens common to PJI and FRI in both planktonic and biofilm phenotypes. However, there were some differences, β-TCP/CS beads loaded with antibiotics displayed reduced potency against P. aeruginosa PAO1 after 4 days compared to CS and potency only extended to 6 days. This relative difference in tobramycin potency may be related to the amount of CS in the material (100% vs 50%) which is more absorbable and has a lower porosity than beta-tricalcium phosphate36 Another possibility is that P. aeruginosa may produce metabolites which interact with the materials in different ways thus influencing potency. This phenomenon is likely not general to all Gram negative bacteria since we saw potency of V + T released from CS beads against strains of K. pneumoniae and A. baumannii for 40 days25, similar to that of staphylococcal strains. It is therefore not unreasonable to speculate that antibiotic loaded β-TCP/CS will show greater potency duration against Gram negative strains other than P. aeruginosa. Nevertheless, in our biofilm challenges we saw significant reductions in P. aeruginosa over 3 days of exposure to both bead types. β-TCP/CS or CS beads mixed with V + T and V + G resulted in completed killing of P. aeruginosa PAO1 at days 1 and 3, demonstrating an in vitro prophylactic effect against biofilm formation. This outcome was similar to our previous study25, which also revealed that combinations of V + T loaded by CS could prevent the PAO1 biofilm formation for at least 3 days. As for S. aureus, both β-TCP/CS and CS beads containing V + T were also able to prevent SAP231 biofilm formation at days 1 and 3, while V + G showed complete prevention at day1 but allowed some growth after 3 days. It is important to note that this assay represents 3 heavy inoculations as well as removal of any antibiotics that might have built up over the proceeding 24 h and thus can be considered a robust challenge.In vitro assays are useful to assess antibiotic compatibility, potency and release kinetics from PMMA as well as other mineral based bone void fillers used in orthopaedic surgery for infection control in PJI and FRI, in the context of managing bacterial biofilms, a complicating factor in these infections. Previously, we have used in vitro methods to demonstrate potency and duration of release of a wide variety of antibiotics from CS beads against planktonic and biofilm cultures37. β-TCP also shows a good biocompatibility38 and faster osseointegration with restoration of physiological architecture compared to HA40. The β-TCP/CS has both bioactive and biphasic properties, with CS acting as a barrier to prevent initial in-growth of the soft tissue and β-TCP acting as a longer term scaffold. The negatively charged surface can stimulate the osteogenic activity, accelerate bone formation and fusion by harnessing key proteins, and direct osteoprogenitor cell proliferation and osteoblast adhesion for rapid osteogenesis41. Our data suggests that β-TCP/CS may also have functionality for releasing antibiotics locally in high enough concentrations and for long enough periods to significantly prevent or reduce existing biofilms as well as providing a longer term osteoconductive scaffold. However, it is also important to consider long term exposure of antibiotics on the development of resistance, particularly since P. aeruginosa is known to develop resistance against aminoglycosides42. There is debate regarding the use of high concentrations of antibiotics in the joint space, on one hand it is argued that if concentrations are high enough for long enough where biofilms are present all bacteria will be killed then there is no chance of developing resistance and there is a greater chance when relying on systemic administration alone where local concentrations are likely not going to reach levels where biofilms can be eradicated43. On the other hand there is not enough clinical data to assess the benefit of locally high concentrations in light of the fear of antimicrobial resistance (AMR)44. In ongoing experiments we are assessing the probability of the development of AMR of antibiotics alone and in combination released from β-TCP/CS and CS beads against our test strains. Also in future work it is important to assess the impact that antibiotics might have on the absorption kinetics with respect to the duration of stability of the scaffold.Although both formulations demonstrated similar potency and antimicrobial activity against biofilms some differences might be explained to differences in mineral chemical and physical compositions. Bone graft substitute, β-TCP and CS (β-TCP/CS) has a finer microarchitecture than CS such that the porosity more closely resembles cancellous bone, thus allowing more rapid absorption than hydroxyapatite (HA)In summary, the present study demonstrated that vancomycin, tobramycin and gentamicin retain potency when mixed and set in β-TCP/CS and release these antibiotics over periods of up to 40 days. Moreover, β-TCP/CS displayed similar efficacy with CS beads in both prevention and significantly reducing bioburden of PAO1 and SAP231 biofilms, suggesting that this material when loaded with antibiotics has potential to stimulate osteogenesis through acting as a scaffold and simultaneously protecting against biofilm infection. However, future in vivo experiments and clinical investigations are warranted to more comprehensively evaluate this use of β-TCP/CS in the management of orthopaedic infections.Supplementary Tables.


Honey bees and other pollinators are threatened by numerous stressors, including virus infections. Currently, no effective treatments are available, stressing the importance of natural defenses. These defenses may be enhanced through selective breeding. This study sought to evaluate the potential for breeding, while also testing a few potential mechanisms of natural immune responses and assessing how widespread viruses are in commercial honey bee queens in the U.S. We identified significant differences in survival of virus infection among and within U.S honey bee stocks, indicating that selective breeding may be able to decrease the virus susceptibility of honey bees. Survival differences may be related to differences in the natural immune system of honey bees and could relate to how much virus stress bees have experienced in the past.Apis mellifera), Israeli acute paralysis virus (IAPV) can be linked to severe honey bee health problems. Breeding for virus resistance may improve honey bee health. To evaluate the potential for this approach, we compared the survival of IAPV infection among stocks from the U.S. We complemented the survival analysis with a survey of existing viruses in these stocks and assessing constitutive and induced expression of immune genes. Worker offspring from selected queens in a common apiary were inoculated with IAPV by topical applications after emergence to assess subsequent survival. Differences among stocks were small compared to variation within stocks, indicating the potential for improving honey bee survival of virus infections in all stocks. A positive relation between worker survival and virus load among stocks further suggested that honey bees may be able to adapt to better cope with viruses, while our molecular studies indicate that toll-6 may be related to survival differences among virus-infected worker bees. Together, these findings highlight the importance of viruses in queen breeding operations and provide a promising starting point for the quest to improve honey bee health by selectively breeding stock to be better able to survive virus infections.Among numerous viruses that infect honey bees ( Apis mellifera) is the world’s most important managed pollinator for agricultural crops and also pollinates a number of natural flowers [Varroa mite and a range of viruses have particularly profound impacts on individual and colony health [The Western honey bee , POL-line (POL), Russian (RUS) bees, as well as “Italians” from California (ITC) and Georgia (ITG) and “Carniolans” from Hawaii (CAH). These stocks were selected to represent a wide variety of commercially available American stocks . While t®  overnight to allow for tissue collection without RNA degradation for subsequent analyses. Each queen was pinned onto a sterile dissection dish and the head, thorax, gut, spermatheca, and ovaries were isolated and frozen until further processing for screening of virus titers (see below). While the queens may differ in virus content from their workers [Upon arrival, four queens from each source were directly sacrificed and frozen at −80 °C. Prior to dissections, these queens were soaked at −20 °C in RNAlaterICE workers , vertica workers ), and quThe remaining six queens from each source were marked on their thorax with different colors to represent their sources and introduced into 3-frame mating nucs containing 500–700 worker bees from common source colonies in an isolated apiary maintained at the University of North Carolina at Greensboro, NC. Colonies were inspected every other day and received sugar patties when needed as supplemental food and newly emerged workers were added to maintain colony size. No Varroa or other disease treatments were necessary during the experimental period. Upon completion of the 21-day brood cycle, frames of emerging brood from each colony were collected and transferred into an emergence incubator . Approximately 100–500 newly emerged workers were kept on their frames until 3–5 days of age and then subjected to IAPV inoculation or a sham treatment. Around ten untreated bees were frozen at −80 °C for each trial as a pre-treatment reference sample.2 for 10–12 s, the thoracic hairs were shaved off and individuals either inoculated by applying an aqueous solution of purified IAPV particles onto their thorax [7 genome copies of IAPV per microliter, less than 103 copies of DWV and Sacbrood virus (SBV), and no measurable Acute bee paralysis virus (ABPV) or Black queen cell virus (BQCV).After anaesthetization with COr thorax  or receir thorax . Cups ofr thorax . Furtherr thorax . The IAPrelS values positive. Absolute and relative survival was compared among stocks and colonies with a hierarchical (2-level) survival analysis using Cox proportional hazard models with mixed effects in “R”, using the “survival” package v. 3.1–12.Data for each colony and treatment were pooled across cups and trials after excluding individual deaths in the first eight-hour interval  and cups that exhibited >50% mortality in any given time interval . To assess the overall effect of IAPV-inoculation vs. sham treatment, a survival comparison between the two groups across all colonies was performed with a log-rank test (Kaplan–Meier module in SPSS v.20 (IBM). After survival of the sham treatment proved significantly different among colonies but not stocks, we continued our survival analysis with absolute survival times of individuals after IAPV inoculation  protocol . The cont value of 35 or lower was recorded.Viruses were quantified in all samples with successful cDNA product from the initially surveyed queens. In addition, viruses were quantified in the pretreatment worker groups. qRT-PCR was performed using primers specific for the following viruses: Black queen cell virus (BQCV), Deformed wing virus , Israeli acute paralysis virus (IAPV), and Kashmir bee virus (KBV), Sacbrood virus (SBV), Acute bee paralysis virus (ABPV), and Chronic bee paralysis virus (CBPV)  and 2–3 days after IAPV-inoculations  were included. The following antimicrobial peptides (AMPs) and other immune genes were selected as targets based on previous studies: dicer-like, argonaute (AGO-2), toll, hopscotch, apidaecin, and hymenoptaecin . Overall virus prevalence was significantly different among stocks only in the gut  but not in the thorax  or ovary .The RT-qPCR surveys of thorax, gut, and ovaries of queens from the six stocks revealed the presence of numerous viruses. The overall virus prevalence  across all body parts was significantly different among stocks , CBPV , BQCV , DWV-B , and DWV-A  were significantly different among stocks across all tissues. When analyzing the quantitative viral loads of each tissue separately, significant differences were found in the thorax for KBV  = 3.5, p = 0.035), CBPV  = 20.7, p < 0.001), and BQCV  = 4.7, p = 0.013), in the gut for BQCV  = 11.8, p < 0.001), DWV-A  = 7.8, p = 0.002), DWV-B  = 3.4, p = 0.041), and DWV-C  = 3.4, p = 0.040), and in the ovary for DWV-B  = 7.7, p = 0.002) and DWV-C  = 13.7, p < 0.001).Analyzed separately , KBV . Relative survival was also significantly different among stocks  but the differences were not the same .Altogether, the survival of 2027 control-treated and 2030 IAPV-inoculated workers was assessed to study differences between stocks and colonies. IAPV-inoculated worker survival was significantly different among stocks . IAPV survival was not significantly correlated to constitutive or induced expression of any other candidate gene.To test whether the pre-experimental expression levels of several immune genes were correlated with worker survival of IAPV exposure, their average ∆CT values were correlated with the average survival of IAPV-inoculated workers  of each colony. Toll-6 expression showed a significant correlation to IAPV survival across colonies . However, when the two components of “relative survival” were correlated to BQCV quantity, it became clear that this negative relation was mainly due to a positive correlation of control survival  and not an effect of BQCV on survival of IAPV-inoculated workers . None of the other viruses  found in the pretreated workers were significantly related to survival differences among colonies. While relative survival was not significantly correlated to the change in IAPV titers {∆∆CT(IAPV)} due to inoculation , survival of the control workers  and of inoculated workers  was significantly correlated with ∆∆CT(IAPV).No evidence of CBPV, KBV, and DWV-C was found in the pretreatment workers. Among the remaining viruses, relative survival of IAPV was negatively correlated to the quantity of BQCV in pretreated workers across colonies (ROur study was designed to evaluate differences in survival of virus infection among worker honey bees from different stocks and test for a relation between survival and viral loads in the respective source populations. We found significant differences in viral loads among queens tested from different stocks. We also identified some survival differences of IAPV infection among stocks, although variability within stocks was much greater. The survival differences among stocks and among colonies within stocks might indicate variation in virus resistance and thus provide a basis for selective breeding to enhance virus resistance in all stocks. Measures of candidate gene expression and other viruses indicated that survival differences after IAPV inoculation may be in part explained by Toll-6, an important immune gene, but more mechanistic follow-up studies are necessary to elucidate the genetic basis of virus resistance in honey bees.Our survey of young queens that we acquired from different sources was limited by a relatively small number of successfully evaluated individuals and tissues. This limitation prompted us to evaluate the prevalence of viruses across all samples as measure of pathogen pressure in the source populations, even though different tissues of the same individual cannot be considered strictly independent of each other . When anImportantly, the virus loads experienced by the different stocks in our study correlated with the differences in IAPV survival. Although our analysis was severely hampered by small sample size, these results suggest that honey bees from high virus environments may have evolved mechanisms to ensure better survival of an acute virus infection. If confirmed, these findings suggest that natural selection is already operating to improve the health of honey bees , at leasLarge sample sizes are required for comparative survival analysis and we measured thousands of individuals. However, the number of individual colonies per stock was limited by absconding of experimental hives or insufficient brood production. Originally planning to analyze the survival of IAPV inoculated workers relative to survival of control workers from each colony, we focused our analyses on the absolute data on IAPV-inoculated worker survival because we found unanticipated differences in survival of sham-treated workers among colonies. To a large extent, these differences could be attributed to a pre-existing infection with BQCV that correlated with survival of control-treated but not of IAPV-inoculated workers. This strong correlation of caged survival with pre-existing BQCV titers is noteworthy because BQCV usually is reported as not harmful to adult workers ,71.In the absence of significant effects of resident viruses and other confounding factors, our IAPV-inoculated worker survival differences among and within stocks can be interpreted as genetic differences in IAPV susceptibility, which has also been reported in the egg stage . We chosWe cannot exclude the possibility that topical applications resulted in some variability of IAPV entering the bees’ body but IAPV titers of inoculated bees were consistently high across all colonies. Additionally, DWV titers were assessed in IAPV-inoculated samples to test for an indirect effect of the inoculation on DWV because previous results indicated that wounding trauma can increase DWV titers . HoweverOur evaluation of six candidate immune genes suggested that the toll-6 pathway might play a role in the identified differences in IAPV susceptibility: Constitutive expression and expression changes were correlated with IAPV-inoculated worker survival across colonies. However, the expression of toll-6 before inoculation was negatively related to survival, contrary to our expectation. Higher constitutive toll-6 expression could have been related to lower survival because it may have indicated an undetected immune challenge prior to infection. Meanwhile, the stronger induction from low initial expression levels to relatively high levels after IAPV-inoculation was related to higher survival. These findings have to be interpreted cautiously due to our small sample sizes, but toll-signaling has been generally implicated in honey bee responses to viral infection , althougWe report standing genetic variation in virus susceptibility among and within different stocks in the US and tentatively relate this variation to possible explanations, such as toll-6 expression and the viral load of the source populations. Additionally, our findings indicate that viruses may be quite common in queens without clear symptoms. Thus, commercial distribution of queens constitutes an effective transmission pathway for viruses to reach new operations at a large spatial scale. We suggest that queen breeders may want to include virus screening into their management and breeding practices. Although our bioassays using individual inoculation are labor-intensive, new methods could be developed to assess virus-susceptibility more efficiently.


Grapevine leafroll-associated virus 3 (GLRaV-3), remain poorly understood. We show that the vine mealybug, Planococcus ficus, feeds through a membrane feeding system on GLRaV-3 viral purifications from both V. vinifera and N. benthamiana and transmits the virus to test plants from plants from both species. Building on this strategy, we used an immunofluorescence approach to localize virions to two retention sites in P. ficus mouthparts. Assays testing molecules capable of blocking virus transmission demonstrated that GLRaV-3-transmission by P. ficus could be disrupted. Our results indicate that our membrane feeding system and transmission-blocking assays are a valid approach and can be used to screen other candidate blocking molecules.Grapevine leafroll disease is one of the most important virus diseases of grapevines and occurs in every major grape-growing region of the world. The vector-transmission mechanisms of the causative agent,  Insect vectors play an essential role in the dissemination of many viruses that cause diseases in humans, animals, and plants. Vector-transmission is a complex event in the virus disease cycle and can be divided into virus acquisition, retention, and inoculation processes. Most plant viruses rely on vectors for efficient transmission, such as fungi, nematodes, and arthropods . InsectsCauliflower mosaic virus (CaMV) ). GLRaV-3 . GLRaV-3 GLRaV-3 ,16. Whol GLRaV-3 . Three re tissue . One-stee tissue . The priPotential virion retention sites in mealybug vectors were localized following the protocol previously described . For artv/v) with expected affinity to substrates on the cuticle of insect vectors [v/v), a molecule commonly used to block nonspecific binding of proteins. Mealybugs were then moved to an artificial diet containing viral purifications from grapevines as described above for another 12 h, followed by manual placement onto test V. vinifera for a 4-day IAP. Petiole samples were collected from plants 4 months post-inoculation, and RNA extractions and RT–PCR were completed as described above. Logistic regression with Firth’s bias correction was used because no acquisition or inoculation occurred in the sucrose treatment resulting in all zeroes (quasi-complete separation of factor levels) [Five to ten mealybugs were placed in feeding chambers, and the opening was covered by a layer of thinly stretched parafilm as described above. The feeding chambers were then placed, parafilm side down, into a small dish containing either artificial diet with a competitor molecule or just artificial diet for a 12-h AAP. The competitor molecules chosen were the lectin wheat germ agglutinin (0.1%  vectors  and case levels) . Analyse levels) .P. ficus could transmit GLRaV-3 from artificial diets to test plants, experiments with virus purified from both V. vinifera and P1/HC-Pro N. benthamiana were conducted  or recipient  plants did not have a significant effect on transmission rates. The interaction within the same plant species transmission was significant when compared to interactions between the two different species . All trials, including P. ficus feeding on artificial diet without purified virus as controls, resulted in no transmission.To determine if onducted . Our resP. ficus were sequentially fed on separate artificial liquid diets containing first, purified GLRaV-3 virions, second, anti-GLRaV-3 IgG, and third, a diet with goat anti-rabbit IgG conjugated with Alexa Fluor 488. A final short wash step on artificial diet alone was included to reduce any nonspecific binding. The same experiment was repeated, placing P. ficus on GLRaV-3-infected grapevines instead of viral purifications during the first step, followed by subsequent diets as described above. All trials included P. ficus feeding on artificial diet without purified virus or on healthy plants as controls. No fluorescent labeling was observed in the control experiments. Our results show that GLRaV-3 was retained in two binding sites in insect mouthparts   on the location of the signal observed, and there was no significant difference in whether the signal was located in the stylet or cibarium  of insects observed.uthparts . When fee labium . Fluorese labium c; in onee labium . No labeP. ficus in artificial diets, followed by feeding on purified virus and subsequent inoculation on V. vinifera (three biologic replicates) to determine if transmission could be disrupted. The competitor molecules tested were the lectin wheat germ agglutinin (WGA) with expected affinity to substrates on the cuticle of insect vectors and casein, a molecule commonly used to block nonspecific binding of proteins in immunoassays. Our results show that WGA significantly blocked GLRaV-3-vector transmission to plants (0/45 tested) in comparison to the sucrose controls (9/46) . Four of 45 plants were GLRaV-3-positive when blocked with casein, with no significant difference when compared to sucrose controls (9/46) . As a control, experiments with P. ficus fed on sucrose instead of a blocking molecule resulted in 9 of 46 plants infected with GLRaV-3. This provides evidence that the virus binds to P. ficus mouthparts and suggests that there is a receptor implicated in virus retention. This assay also provides proof of concept that this approach is valid for testing other compounds capable of blocking transmission.Because GLRaV-3 appears to be retained in one or two binding sites, further questions remain regarding the nature of the receptors involved in the mouthparts. Virus transmission experiments were conducted by providing a blocking compound to N. benthamiana [N. benthamiana and V. vinifera could be transmitted through an artificial diet membrane system to healthy plants. The purification protocol used was based on a protocol previously described for LIYV, a related long filamentous virus [GLRaV-3 is one of the most important viruses of grapevines, but there are significant gaps in our understanding of its transmission biology. GLRaV-3 research has been limited by a labor-intensive and technically challenging host–pathogen system until the recent discovery that GLRaV-3 is capable of infecting an alternative model host, thamiana . Buildinus virus . As deteus virus .V. vinifera purifications to V. vinifera plants compared to transmission to P1/HC-Pro N. benthamiana or from P1/HC-Pro N. benthamiana purifications to V. vinifera or P1/HC-Pro N. benthamiana plants. It is possible that transmission could be affected by P. ficus host plant preference. Observations of P. ficus behavior on V. vinifera versus N. benthamiana have been previously described and demonstrated that P. ficus prefers V. vinifera [N. benthamiana purifications; P. ficus may reject N. benthamiana as a recipient host, or there is some component purified from N. benthamiana that deters P. ficus feeding on diets. It also may be that P. ficus prefers probing plants instead of parafilm, as the transmission rates published in earlier work from N. benthamiana or V. vinifera plants to both N. benthamiana and V. vinifera plants were higher [N. benthamiana. Furthermore, while this work demonstrates that artificial diets may be used to study mealybug–ampelovirus interactions and that the protocol used for virus purification yields intact particles, methodological improvements would allow for a range of additional hypotheses to be tested.GLRaV-3-transmission was highest from vinifera . In these higher . Lastly,P. ficus transmitted GLRaV-3 from artificial diets through membrane feeding, a unique immunofluorescent localization system previously used to investigate LIYV transmission in whitefly vectors was adapted for this study. This approach proved to have low efficiency in this host–pathogen system compared to the results observed in whiteflies, with 31 out of 645 first-instar P. ficus observed with a fluorescent signal  of P. ficus as well as on the retracted stylet tips, regardless of the initial source diet . In one insect, a signal was observed in both of these regions at the same time. It is possible that the virus is retained in both sites, but further studies testing other GLRaV-3 variants and mealybug vector species should confirm if transmission of the virus is associated with one or both of the sites. Without associated transmission data, it cannot be concluded whether the retention sites observed are implicated in the transmission of GLRaV-3.Because t signal . A similications . In ordeMaize chlorotic dwarf virus, aphid-transmitted Anthriscus yellows virus, and Parsnip yellow fleck virus have also been localized to the tips of stylets or foreguts of insect vectors [Cucumber mosaic virus and other potyviruses [Although it cannot be confirmed which site is associated with transmission from this work, it is encouraging that these results follow trends observed in other virus–vector systems. Both the stylet tips and cibarium regions have been implicated in nonpersistent or semipersistent virus transmission. The retention site of the related noncirculative LIYV has recently been identified in the cibarium of the whitefly vector . Retenti vectors ,22,23. Tyviruses ,24.Citrus tristeza virus, a member of the Closteroviridae family, virions were shown to bind to the N-acetylglucosamine moieties of the cuticular surface of the aphid cibarium [Xylella fastidiosa, a noncirculative bacterial pathogen that colonizes the foregut of leafhopper vectors [Crinivirus LIYV and both the CPm and heat shock proteins in Citrus tristeza virus, an aphid-transmitted Closterovirus, are viral proteins required for successful retention and transmission by insect vectors [In an effort to further characterize the nature of virus-vector interactions, we tested if the transmission of GLRaV-3 could be disrupted by either casein or the lectin wheat germ agglutinin (WGA) . Our rescibarium . Further vectors ,25. This vectors ,7. Furth vectors .P. ficus mouthparts. Although the viral proteins required for binding remain unknown, vector transmission was blocked after binding of WGA, suggesting that the virus interacts with the cuticular surface of the mouthparts. This provides first insights into the transmission biology of this economically important host–pathogen system and demonstrates that mealybug feeding through artificial diet systems works for future studies. Further investigations are needed to elucidate the specific viral proteins required for transmission. The creation of recombinant viral proteins and specific antibodies could help confirm the viral retention site and transmission strategies of this system.In conclusion, our analyses of GLRaV-3-transmission mechanisms suggest that viral retention can be narrowed down to one or two binding sites in


All cellular organisms coevolve with multiple viruses, so that both virus-host and intervirus conflicts are major factors of evolution. Accordingly, hosts evolve multiple, elaborate defense systems and viruses respond by evolving means of antidefense. Although less thoroughly characterized, several dedicated mechanisms of intervirus competition have been described as well. Recently, the genomes of some bacterial and archaeal viruses have been shown to harbor CRISPR mini-arrays that typically contain a single spacer targeting a closely related virus. The involvement of mini-arrays in an intervirus conflict has been experimentally demonstrated for a pair of archaeal viruses. We model the evolution of virus-encoded CRISPR mini-arrays using a game theoretical approach. Analysis of the model reveals multiple equilibria that include mutual targeting, unidirectional targeting, no targeting, cyclic polymorphism, and bistability. The choice between these evolutionary regimes depends on the model parameters including the coinfection frequency, differential productivity of the conflicting viruses, and the fitness cost of mini-arrays. At high coinfection frequencies, the model becomes a version of the Prisoner’s dilemma in which defection, i.e., mutual targeting between the competing viruses, is the winning strategy. Most microbial communities are associated with highly diverse and abundant viral populations . Althougcas genes. However, repeats in mini-CRISPR arrays are identical to those found in the host’s CRISPR-Cas locus, suggesting compatibility of the viral mini-array with CRISPR-Cas components of the host. In some cases, pairs of viruses infecting the same host target each other through their mini-CRISPR arrays. Such mutual targeting supports the role of mini-CRISPR arrays as weapons in interviral conflicts which, combined with the cas genes from the host, target and destroy other viruses that could coinfect the same host. In the case of proviruses and other non-lytic viruses, mini-CRISPR arrays would constitute heterotypic superinfection exclusion mechanisms that promote long-term survival of the carrier by protecting the host from potentially lethal infections. Recently, intervirus competition among archaeal viruses carrying mini-arrays with cross-targeting spacers has been validated experimentally ; and two temperate viruses or proviruses. Finally, we briefly discuss some extensions of the model to account for the loss of CRISPR spacers and the competition among more than two viruses.A and B, both infecting the same host. Mixed coinfections, in which viruses A and B infect the same cell, are a central ingredient of the model. From the point of view of virus A, the coinfection probability pB—A is the probability that virus A encounters virus B during the infection process. Similarly, we define the coinfection probability for virus B, pA—B, as the probability that virus B encounters A in the course of a single infection. From the perspective of the host population, the virus-centric coinfection probability pB—A can be understood as the conditional probability of finding a host infected by virus B given that the host is also infected by virus A. Applying Bayes’ theorem, we getLet us consider two viruses,pA and pB are the probabilities of finding hosts infected by virus A and virus B, respectively. It is clear from this equation that the coinfection probabilities pB—A and pA—B can take different values. Such differences are caused by differences in abundance, infectivity, spatial distribution, host range, and any other factor that introduces an imbalance in the prevalence of viral infections. All other factors being equal, the coinfection probability will be greater for the virus with the lower abundance, the more restricted distribution, or the narrower host range.where pB|A = pAB|A + pBA|A and pA|B = pAB|B + pBA|B, where the subindices AB and BA indicate the sequence in which infections occurred. We introduce the parameters qA and qB to denote the fractions of the coinfection events in which viruses A and B were, respectively, the first to arrive. From these definitions, it follows that pAB|A = qA×pB|A and pBA|B = qB×pA|B.In the case of viruses that target each other via mini-CRISPR arrays, the outcome of a coinfection event likely depends on which virus infected the host first. To account for that, we split each coinfection probability into two components, that is, We model the competition among two groups of viruses (species or strains), each of which can possess or lack mini-CRISPR arrays against the other. For simplicity, and without loss of generality, we normalize the productivity of an infection by a single virus that lacks mini-CRISPR arrays to make it equal to 1 . When two viruses coinfect the same host, the offspring of each virus depends on whether they possess or lack mini-CRISPR arrays targeting each other:(a)w . If coinfection does not affect the total yield and both viruses are equally competitive, we can simplify wA = wB = 1/2. By using different values of wA and wB, the model can account for unequal competitive abilities (wA≠wB), interference (wA + wB < 1) and facilitation (wA + wB > 1) between viruses.In the absence of such arrays, the average productivity of each virus in a mixed coinfection is given by the parameter (b)1-c for the virus that carries the array and 0 for the targeted virus. Parameter c (denoted as cA for virus A and cB for virus B) is the cost of having a mini-CRISPR array with a spacer that matches a sequence that is often closely similar to a sequence in the genome of the mini-array-carrying virus itself .If only one virus has a mini-CRISPR array with a spacer against the other virus, the productivities are (c)q(1−c). In general, q and c are different for viruses A  and B . As explained above, qA can be interpreted as the probability that virus A arrives first and virus B arrives later (and the reverse for qB). Note, however, that the “first who arrives wins” assumption can be easily relaxed to allow for some degree of mutual destruction upon coinfection, in which case qA + qB < 1.If both viruses target each other, we assume that “the first that arrives wins.” This is biologically plausible because, whereas transcription and processing of mini-CRISPR arrays require some time, scanning and cleavage of the targeted viral DNA is very fast . TherefoA and B can be modeled as an evolutionary game, with the strategies being having or not having a spacer against the other virus. The fitness values of each virus are given in A and B are homogeneous with respect to their strategies .The parameters of the model are summarized in B lacks anti-A spacers, the fitness of virus A is given by the second column of A benefits from anti-B spacers if pB|A > cA/(1−wA). Conversely, if the entire population of virus B carries anti-A spacers (first column of A will benefit from anti-B spacers if pB|A > cA/(qA−qAcA + cA). Thus, the outcome of the evolutionary process is determined by a trade-off between the cost of the CRISPR mini-array and the potential benefit conferred by the spacer against the competing virus during coinfections.Before proceeding with the formal analysis of the system, some immediate insight can be gained from inspecting the fitness matrix of the evolutionary game. If we consider a scenario in which virus xA and xB, both with values between 0 and 1, which represent the fraction of viruses of each strain that harbor CRISPR spacers against the other strain. Conversely, 1−xA and 1−xB are the relative sizes of the spacer-free subpopulations. Focusing on strain A, the fitness of viruses with and without anti-B spacers  results from multiplying the fitness matrix in B that harbor and lack anti-A spacers:To analyze the general case of mixed populations, let us introduce new variables Because of the complexity of virus-host interaction networks in natural microbial communities, it is unlikely that the host ranges or geographical distributions of two viruses completely overlap . The exiA is the mean fitness of strain A, calculated aswhere ϕB) and class–specific fitness values  for strain B are obtained by exchanging subindices A and B in Eqs 3 and 1, respectively. After some manipulation, the equations that describe the population dynamics can be rewritten in a more informative form asThe mean fitness . An intuitive interpretation of these critical thresholds is that they represent the coinfection probabilities for which the benefit of targeting the competitor virus outweighs the cost of the CRIPSR array, either when the competitor lacks a cross-targeting spacer and hence does not engage in the arms race (P∗) or when it does (P∗∗). Depending on the coinfection rate, the evolutionary arms race can lead to 5 qualitatively different regimes ((note that we omit the subindices  regimes :(a)No targeting if (b)A targeting B if Unidirectional targeting, with (c)B targeting A if Unidirectional targeting, with (d)Mutual targeting if (e)pB—A <pA—B <pB—A <pA—B <Cyclic dominance, with an alternation of targeting and non-targeting subpopulations, if A or B) either possess or lack the mini-CRISPR array. Second, for some parameter combinations, the equilibrium is not unique, that is, the system displays bistability. Specifically, if pB—A <pA—B <A to B or from B to A , will evolve. Which of the two possible equilibria is reached in that bistable regime, depends on which virus acquired the spacer first. The existence of this regime implies that instances of unidirectional targeting can be found even if viruses A and B are identical in all their parameters. Alternatively, if pB—A <pA—B <Two important observations are pertinent. First, with the exception of the last regime, the population always reaches an equilibrium state in which all viruses of a given type (wA) because those would benefit the most from curtailing other, more efficient coinfecting strains.It follows from Eq. 9 that, in order to maintain a mini-CRISPR array, the probability of coinfection must be greater than the cost of the array. Moreover, possession of mini-CRISPR arrays is more advantageous for strains with low yields in mixed coinfections . Similarly, unless one of the viruses has specialized to exploit the other (which is unlikely given the close relatedness), similar coinfection yields (wA = wB = w) can be expected. With these simplifications, the threshold coinfection probabilities required for the maintenance of mini-CRISPR arrays (Eqs 9–10) become:When considering 2 lytic viruses, the model can be simplified by noting that any of them can be the first to enter the host cell with the same probability, that is,  related . Therefow≈1). Furthermore, the greater the degree of interference , the lower the critical coinfection threshold.The dependencies of these thresholds on the fitness cost and the productivity in mixed coinfections are plotted in w≈1/2). If we also assume that the cost of the array is small (c≪1), the critical coinfection probabilities in Eqs 11–12 tend to the same value, P∗∗≈P∗ = 2c. The possible evolutionary outcomes under this scenario become much simpler, with each virus maintaining a mini-CRISPR array against the other virus only if its coinfection probability is greater than twice the cost of the array (w < 1/2) introduces additional outcomes and broadens the range of conditions in which unidirectional targeting can evolve  and one temperate (T) virus. By temperate virus, we refer both to proviruses integrated in the host genome and to non-lytic viruses that produce non-lethal chronic infections. Although the latter are particularly common in archaea . Once again, P∗ and P∗∗ represent the critical coinfection probabilities beyond which it becomes profitable to target the other virus that either does (P∗∗) or does not engage (P∗) in the arms race via its mini-CRISPR array.. In terms of the model, this implies that temperate viruses can maintain mini-arrays against lytic viruses as long as the coinfection probability is greater than the cost of the mini-array (cT). Thus, frequent encounters between the temperate and the lytic virus will result in unidirectional targeting from the former toward the latter. The same reasoning applies to those proviruses that do not provide immunity against a given lytic virus. However, many proviruses encode superinfection exclusion mechanisms that prevent infection by closely related viruses and, sometimes, even by distantly related ones , and leads to a cyclic targeting dynamics  if the coinfection probabilities for both viruses are greater than the cost of the mini-array .The outcome of the competition between a lytic and a temperate virus depends on (i) whether the latter is an actively replicating non-lytic virus or a provirus. For a provirus, we must distinguish (ii) whether or not it provides the host with CRISPR-independent immunity against subsequent infection by the lytic virus, and (iii) whether such immunity can be overcome by a lytic virus that carries a mini-array against the temperate virus. Let us start with the case of an actively replicating non-lytic virus. Because the long-term productivity of chronic viruses is tightly linked to the survival of the host, superinfection by lytic viruses severely reduces their fitness (ted ones . Some ofted ones  but it iA) infects a cell that already hosts another temperate virus or provirus (B). Unlike in the previous section, which dealt with viruses that qualitatively differ in their lifestyles, here, the reverse scenario in which B infects a cell that already hosts A is also possible and, once the population reaches equilibrium, equally likely. Accordingly, to apply the general model to 2 temperate viruses, we set the parameter q = 1/2. Additionally, to study competition among temperate viruses, it is necessary to distinguish between the cases of actively replicating non-lytic viruses and proviruses.Let us consider a scenario in which a temperate virus (A when it targets virus B becomes (1-cA)(1-pB|A + qApB|A), regardless of whether virus B targets A or not. With this modification, after substituting q = 1/2, the critical threshold P∗ for the competition of 2 proviruses becomesThe model for the competition between two actively replicating temperate viruses is formally the same as for two lytic viruses, with the critical coinfection thresholds given by Eqs 11–12 . By contP∗∗ remains the same as in Eq. 12.whereas w≥1/2), the possible evolutionary outcomes would be those shown in P∗∗≈2c and P∗≥1. Under this assumption, we would expect either no targeting or mutual targeting between proviruses , with mutual targeting evolving only if the coinfection probability of both viruses is greater than twice the cost of the mini-array. The same outcomes are predicted if one or both proviruses possess superinfection exclusion mechanisms that can be overcome through CRISPR-mediated targeting . In contrast, if superinfection exclusion occurs and cannot be counteracted by CRISPR-mediated targeting, the benefit of harboring the mini-array vanishes for every provirus that encodes superinfection exclusion mechanisms .For both proviruses and actively replicating non-lytic viruses, the possible evolutionary outcomes will depend on the effect of coinfection for the long-term productivity of the temperate virus. In the case of actively replicating non-lytic viruses, it seems reasonable to assume that coinfection substantially decreases viral productivity. To get an approximate idea of how that affects the evolutionary outcome, if the productivity of the resident virus decreases to half of its original value, the evolutionary regimes will coincide with those shown for lytic viruses in In the model discussed so far, the only factor that opposes the engagement of viruses into CRISPR-mediated arms races is the fitness cost of mini-arrays. Motivated by the fact that prokaryotic genome evolution displays a consistent deletion bias  and by sd, Eq. 2 becomes a replicator-mutator equation:Mini-array loss can be easily incorporated into the present framework as follows. If spacers are lost at a fixed rate fA,1, fA,2, fB,1, fB,2, ϕA, ϕB as before. For simplicity, we present the equations for w = 1/2 and omit subindices unless necessary to prevent ambiguity. The first critical coinfection threshold becomeswith the same fitness parameters The general expression for the second critical threshold becomes more complicated because it indirectly depends, for a given virus, on the coinfection probability for the other virus. Specifically,wheresB is the relative reduction in the fitness of virus B associated with the loss of the CRISPR mini-array , provided that virus A does not engage in the arms race. Similar expressions are obtained for sA by replacing subindices A and B. Due to their dependency on pA—B and pB—A, the critical coinfection thresholds pA—B vs. pB—A parameter space. Nevertheless, the parameter combinations that lead to no targeting, unidirectional targeting, and mutual targeting can still be obtained by applying the rules from d≪sA, d≪sB), q)(1−c)(1−d) > 1/2 and q)(1−c)(1−d) < 1/2). For larger values of the loss rate, the bistable regimes are determined by a more convoluted set of equations (not shown here), and the range of parameters that allow for bistability shrinks as the loss rate grows.In these expressions, c=0), and spacer loss is the only force counteracting selection for the mini-CRISPR array. To further simplify the final expressions, we assume that the loss rate is small compared to the strength of selection . These considerations lead to the critical thresholds P∗ = 2d and P∗∗ = d/(q−qd + d), which coincide with the expressions in the original model (Eqs 9–10) but with the cost of the array replaced by the loss rate.The expressions from Eqs 18–20 fully apply to a loss-driven scenario where the mini-CRISPR array entails no cost  the cost of maintaining a mini-CRISPR array with the virus-targeting spacer, and (ii) the productivity of a virus lacking a mini-CRISPR array in a mixed coinfection relative to the productivity of that same virus in isolation. For two lytic viruses, a necessary (and often sufficient) condition for the emergence of mutual targeting is that the coinfection probability is greater than twice the cost of having the array. The same condition holds for the emergence of mutual targeting between two temperate viruses (either proviruses or actively replicating non-lytic viruses). For the competition between lytic and temperate viruses, the model predicts evolution of unidirectional targeting, from the temperate virus to the lytic virus, or cyclic targeting dynamics if the coinfection probability exceeds the cost of the mini-array. Mutual targeting between lytic and temperate viruses is unstable because lytic viruses only benefit from targeting a temperate virus if the latter does not already target the lytic virus. These results emphasize the interplay between superinfection exclusion and CRISPR-mediated targeting in interviral conflicts that involve prophages. Indeed, the outcome of the mini-CRISPR array-mediated arms race critically depends on whether the competing viruses encode mechanisms to prevent superinfection and whether such mechanisms can be vanquished by viruses that carry mini-CRISPR arrays.Our analysis sheds light on the effect of viral interference and facilitation on interviral conflicts, and predicts that mini-CRISPR arrays are more likely to evolve in pairs of closely related viruses that strongly interfere with each other when coinfecting the same host cell. Another prediction that arises from the model is that mini-CRISPR arrays should be more frequent in “specialist” viruses  than in “generalist” ones, especially if the virus-host interaction network is nested as suggested by empirical data . The reaE. coli, they would imply that the CRISPR-mediated arms race among closely related viruses can only be supported if coinfections with a competing virus represent a substantial fraction (about 10% or greater) of the infections by a given virus, or if spacers are divergent enough from the homologous sequence in the virus’ own genome to avoid primed adaptation. An alternative possibility is that viruses that employ CRISPR arrays to target competitors possess mechanisms to inhibit primed adaptation, as has been suggested for bacteriophage T7  with high sequence identity in their protospacer regions . As a rephage T7 . In the A encounters virus B more frequently than B encounters A , it is more likely that only A would carry a spacer against B. Other causes that could lead to asymmetric outcomes would be unequal fitness costs and different yields in coinfection . Nevertheless, the fact that the spacers in CRISPR mini-arrays typically target closely related viruses and the observed instances of mutual targeting strongly suggest that the main role of mini-CRISPR arrays is related to interviral conflict.There could be additional potential benefits that mini-CRISPR arrays might provide to the carrier virus and that are independent of interviral conflict but rather have to do with overcoming the host defenses. For example, high levels of transcription of the mini-CRISPR array could potentially lead to competitive exclusion of the host spacers from the CRISPR-Cas effector modules, protecting the virus from targeting. Additionally, repeats in the mini-CRISPR array could promote the insertion of the virus within the host CRISPR array, thus impairing the host immune response; indeed, insertion of proviruses into CRISPR arrays has been observed in several bacterial genomes . These sFinally, it is worth noting that the modeling framework developed here can be generalized beyond viruses, to plasmids that might carry mini-arrays  and beyoAll datasets for this study are included in the article/supplementary material.JI and GF conceived the study. JI constructed the model. JI, GF, YW, and EK analyzed the results. JI and EK wrote the manuscript that was edited and approved by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mobile health  approaches offer potentially affordable ways to support the care of HIV-infected patients in overstretched health care systems. However, only few studies have analyzed the costs associated with mHealth solutions for HIV care.The aim of this study was to estimate the total incremental costs and incremental cost per beneficiary of an interactive SMS text messaging support intervention within a clinical trial.The Mobile WAChX trial (NCT02400671) evaluates an interactive semiautomated SMS text messaging intervention to improve adherence to antiretroviral therapy and retention in care among peripartum women infected with HIV in Kenya to reduce the mother-to-child transmission of HIV. Women were randomized to receive one-way versus two-way SMS text messages. Messages were sent weekly, and these messages included motivational and educational content and visit reminders; two-way messaging enabled prompt consultation with the nurse as needed. Microcosting methods were used to collect resource-use data related to implementing the Mobile WAChX SMS text messaging intervention. At 2 sites (Nairobi and Western Kenya), we conducted semistructured interviews with health personnel to identify startup and recurrent activities by obtaining information on the personnel, supplies, and equipment. Data on expenditures and prices from project expense reports, administrative records, and published government salary data were included to estimate the total incremental costs. Using a public provider perspective, we estimated incremental unit costs per beneficiary and per contact during 2017.The weighted average annual incremental costs for the two-way SMS text messaging group were US $3725 per facility, US $62 per beneficiary, and US $0.85 per contact to reach 115 beneficiaries. For the one-way SMS text messaging group, the weighted average annual incremental costs were US $2542 per facility, US $41 per beneficiary, and US $0.66 per contact to reach 117 beneficiaries. The largest cost shares were for the personnel: 48.2% (US $1794/US $3725) in two-way and 32.4% (US $825/US $2542) in one-way SMS text messaging groups. Costs associated with software development and communication accounted for 29.9% (US $1872/US $6267) of the costs in both intervention arms .Cost information for budgeting and financial planning is relevant for implementing mHealth interventions in national health plans. Given the proportion of costs related to systems development, it is likely that costs per beneficiary will decline with the scale-up of the interventions. In 2017, an estimated 180,000 children became infected with HIV [Use of SMS text messaging communication is a promising approach for improving ART adherence in peripartum women. Several studies and meta-analyses have shown that regularly delivered SMS text messages can improve ART adherence and retention in care outside of pregnancy -5 and thWhile SMS text messaging interventions are supported by policymakers to improve ART adherence, little is known about the value for money of such technologies. In a global survey conducted by the World Health Organization, lack of evidence on economic evaluation was identified as a major barrier to implementation of mHealth solutions in resource-constrained settings . One of The Mobile WAChX trial (NCT02400671) evaluates one-way versus two-way communication versions of a semiautomated SMS text messaging intervention to improve ART adherence and retention in care in peripartum women at 6 facilities in Nairobi and Western Kenya. One-way SMS text messaging consisted of weekly automated motivational and educational SMS text messages and clinic visit reminders. Participants randomized to the two-way SMS text messaging arm additionally had the capability of communicating with a nurse through the SMS text messaging system. Participants randomized to the control arm received no SMS text messages (standard of care).The study procedures for the randomized controlled trial are described in detail in a previous paper . For cosWe used an activity-based ingredients approach to identify all activities undertaken to deliver the Mobile WAChX project. Activities included intervention planning, project partner sensitization, staff training, system development, and delivery of SMS text messages. After identifying all intervention-related activities, we quantified the resources used and valued these by using the best available data on salaries and commodity prices. We used a combination of data collection methods to collect primary resource-use and cost data, including obtaining prices from project expense reports, administrative records, and published government salaries, and conducting semistructured interviews with facility-based health workers and project administrators. Time-motion studies were conducted in both facilities to record staff time spent on intervention activities . Data collection was conducted between October 2017 and January 2018.We organized cost data into one-time fixed costs and variable costs . Fixed cWe developed an Excel-based model  to estimate total incremental costs and incremental unit costs. The sum of all the activity cost categories reflects all the resources required to deliver the Mobile WAChX intervention. All activities were mutually exclusive, thereby avoiding double counting. We used project output data on the number of beneficiaries per month per facility and number of messages sent by the system and nurses per month. These data were collected as part of the Mobile WAChX monitoring and evaluation strategy. We first estimated the total incremental cost for each facility to deliver the Mobile WAChX intervention and divided this by the number of women receiving the intervention to determine the cost per beneficiary. We also estimated the cost per contact, defined as the total cost divided by the total number of messages sent to participants during 2017, for each facility. We analyzed the costs of one-way and two-way SMS text messaging interventions separately. We also estimated the average weighted costs for both facilities by using project output data on the number of beneficiaries per facility.In addition to estimating intervention costs, we estimated 3 scenarios. The first scenario was to estimate the cost when the two-way Mobile WAChX intervention was implemented in all 6 facilities in this project. In this scenario, the same intervention and personnel were applied to every facility where they shared most start-up costs from system development, cost of system-hosting platform, as well as personnel supervision and coordination costs. The number of participants was the total number of participants receiving intervention in all 6 sites. The second scenario was to estimate a more typical scenario where the MOH of Kenya supports these activities after the pilot phase. We applied the MOH salary scale to service delivery health workers in the current project. Finally, we calculated the incremental cost effectiveness ratios (ICERs) of comparing two-way SMS text messages to no intervention in the 2 facilities. Due to unavailable efficacy data in the Mobile WAChX trial, we used clinical outcome results from a similar randomized controlled trial, which assessed whether two-way SMS text messaging interventions improved plasma HIV-1 viral RNA load suppression at 12 months in 3 clinics in Kenya . The effAmong the 152 HIV-infected women in facility A who received the Mobile WAChX intervention, 76 (50.0%) were randomized to the two-way SMS text messaging group and the other 76 (50.0%) were randomized to the one-way SMS text messaging group. A total of 80 women participated in facility B, of whom 39 were randomized to the two-way SMS text messaging intervention group and 41 were randomized to the one-way SMS text messaging intervention group . The benCost estimates in the 2 scenarios compared to the baseline scenario estimates reflected in The ICERs of comparing two-way SMS text messaging versus no intervention in the 2 facilities and the input parameters are summarized in This is the first study to estimate the costs of an mHealth intervention to promote prevention of MTCT (PMTCT)-ART adherence among peripartum women. Previous studies have shown that SMS text messaging interventions have a positive impact on ART adherence and maternal and neonatal health outcomes in low-and-middle income settings ,17-20. HOnly a few studies have provided the cost estimates of mHealth interventions, including SMS text messaging interventions, in low-income countries. The MAMA program was initiated in 2012 in South Africa to enhance the utilization of MCH services among pregnant and postpartum women by sending registered users SMS text messages twice per week. The estimated program costs over 5 years was US $1.2 million, 17% of which was incurred by costs on program development and 31% on SMS text message delivery costs . The ChiPersonnel costs accounted for the largest share of the total costs in our project , followed by software development of the SMS text messaging management system and communication costs. Our results are consistent with findings from previous studies that labor costs for delivering other SMS text messaging interventions were the main drivers of the total program costs, followed by SMS text messaging program development ,12,22. HOur findings provide important costing information for budgeting and financial planning for implementing mHealth interventions to achieve UHC in Kenya. mHealth interventions, as a part of the broader eHealth interventions, may become transformational strategies in addressing public health challenges and striving toward UHC in Kenya. This need has also been reaffirmed by the Kenya’s Health Policy (2014-2030), National eHealth Policy (2016-2030), Information Technology Master Plan, and the Health Bill -10. WhilTo our knowledge, this is the first study to estimate the costs of an mHealth intervention targeting PMTCT and MCH in Kenya. Our study has limitations. First, we did not include the labor costs of international collaborators who contributed to the program design and installation and other start-up activities such as work planning meetings. Second, we had to make assumptions about the allocation of communication costs to research and implementation activities, which may influence the total implementation costs. Third, we did not allocate equipment costs to research activities, which may lead to overestimation of the total implementation costs. Equipment such as laptops and phones were mainly used for service delivery, and we do not have detailed information on how much equipment was used to support the research-related activities. Therefore, the evidence should be used with the consideration that after taking into account the equipment costs allocated to research activities, the total incremental cost of implementing SMS text messaging interventions would be lower than the estimates in this study.In conclusion, this study fills the knowledge gap on the costs of mHealth approaches for improving PMTCT-ART adherence among pregnant women in Kenya. When operating at scale, there may be opportunities to reduce the costs per beneficiary. As the Mobile WAChX intervention is scaled up, further research is needed to understand the economic impact from different perspectives, including cost-utility analyses to assess the value for money compared with alternative approaches to improve women’s clinical outcomes and adherence to HIV treatment as part of PMTCT services.


Varroa spp. and Nosema spp.Veterinarians are educated in prevention, diagnosis and treatment of diseases in various vertebrate species. As they are familiar with multifactorial health problems in single animals as well as in herd health management, their knowledge and skills can be beneficial for the beekeepers and honeybee health. However, in education and in practice, honeybees are not a common species for most veterinarians and the typical veterinary diagnostic methods such as blood sampling or auscultation are not applicable to the superorganism honeybee. Honeybee colonies may be affected by various biotic and abiotic factors. Among the infectious agents, RNA-viruses build the largest group, causing covert and overt infections in honeybee colonies which may lead to colony losses. Veterinarians could and should play a more substantial role in managing honeybee health—not limited to cases of notifiable diseases and official hygiene controls. This review discusses the veterinary diagnostic approach to adult bee examination with a special focus on diagnosis and differential diagnosis of the common virus diseases Acute Bee Paralysis Virus (ABPV)-Kashmir Bee Virus (KBV)-Israeli Acute Paralysis Virus (IAPV)-Complex, Chronic Bee Paralysis Virus (CBPV) and Deformed Wing Virus (DWV), as well as coinfections like  Apis mellifera L.) is only rarely in contact with veterinarians as a patient. Official veterinarians and diagnostic laboratories are responsible for the analysis of honey samples and for animal disease control in case of, e.g., American foulbrood, but the general disease prevention and control are done by the beekeepers. The majority of the establishments for veterinary education in Europe accredited by ESEVT  offer elective or no training in honeybee veterinary medicine. Only in 24 of 68 surveyed schools honeybee veterinary medicine is part of the curriculum as a separate subject [In contrast to human medicine, one of the most prominent features of veterinary medicine is that a veterinarian has to deal with a large variety of species. Whereas dogs, cats and horses are common patients in a veterinary practice, the honeybee  and sunflower (Helianthus annuus), pollen harvest severely declines [A variety of biotic and abiotic factors have an impact on honeybee colonies. Belsky et al. presented a broad overview of these stressors: habitat and climate changes, weather, the density of apiaries and food resources, as well as transportation of colonies, are external aspects influencing the honeybee . Equallydeclines  and limideclines . Variousdeclines .This review presents basic information on selected virus diseases and the process of colony examination and diagnostics from the veterinary perspective, with a focus on adult honeybees.Viruses, mostly positive single-strand RNA viruses, are the largest class of honeybee infecting pathogens . Over 20Dicistroviridae. During the last years, this complex has been reported in association with colony collapse disorder (CCD), a phenomenon of severe colony weakening without visible worker bee mortality [ABPV, KBV and IAPV are three similar icosahedral viruses of the family ortality ,20,21. Aortality . Hou andortality .Varroa mite plays an important role as a mechanical vector [Transmission of these viruses is possible vertically as well as horizontally. Furthermore, the l vector . It tranl vector .CBPV is an unclassified RNA virus that causes two different syndromes in honeybees. Type A, the paralytic form, is characterized by trembling, disorientation and ataxia. Type B affected bees show black, hairless and greasy shining abdomens. Both can occur in the colony at the same time and lead to massive worker bee losses .Comprehensive information on this virus can be found in our case report presenting an overt infection with CBPV in two colonies .Varroa destructor plays the key role in transmission as well as virulence and pathology of the virus [DWV is a picorna-like virus, that is often involved in winter losses of honeybee colonies. Two main variants, A and B, can be differentiated ,26,27,28he virus . Obvioushe virus . Infectehe virus .As mentioned above, honeybee health is often influenced by many different factors . WithoutFrom the veterinary perspective, a holistic diagnostic work-up of medical issues in honeybee colonies is important, because the environment, the colony, the pathogens and every bee are each just a link in the chain leading to occurrences of infections . Figure Varroa infestation monitored and controlled? Is the beekeeper migrating the colonies? Maybe the beekeeper can also offer information about weather, crops in the region around the apiary or the density of apiaries in the vicinity—information needed for epidemiological evaluation.Like in any other species, clinical examination starts with a detailed anamnesis. Gathering information from the beekeeper gives a first overview about the acute problem, which often is superficial and skewed by incorrectly interpreted data . Asking The location and the environment around the beehive have an immense relevance to the occurrence of health problems and should therefore be carefully observed. The weather, the food range, the landmarks and further factors influence the bees in a similar way as a stable or field does to cattle, for example. A complete hazard analysis is shown by Formato and Smulders . VitallyFirst pathologic findings can be identified during observation of the alighting board and entrance hole. The mass of bees flying in and out—depending on temperature, weather, time of day and state of colony development—provides a first impression about the strength of a colony. Dead bees as well as bees and larvae with alterations of the phenotype, carried out of the hive, may be found in front of the entrance. Veterinarian and beekeeper should take their time to observe the entrance hole carefully A,B. The The “internal” clinical examination of the bee colony and of individual bees are the next steps. It is important to have a look at the adult bees of all castes , as well as the brood in different stages and the hive material. Honeybees are social insects that can survive within the colony as a superorganism only. The colony is functional if each single individual performs its tasks. In the following, we will focus on the clinical findings in adult bees.Varroa mites as well as behavioral, neurological, social or digestive symptoms. Signs of CBPV disease are hairless, black bees and/or neurological symptoms such as trembling, circling or paralysis [Varroa indicate an intense infestation rate with the mite. Phenotype changes, e.g., crippled bees, high mortality and paralysis symptoms, can also be a sign of intoxication. Clinical examination of the bees and the frames should be performed together with the beekeeper. This way, it is possible to analyze the beekeeper´s operations while handling the bees . Generalaralysis ,24. AbnoWhile examining the bees, the frames are assessed: the color and brightness of the wax correspond to the age of the combs. Old wax, having been used in several brood cycles, is much darker than newly produced wax. The frames themselves can be clean or show fecal spots in case of digestive problems . Pollen It is advisable to have a look at how easily the frames can be removed from the hive by the beekeeper. If they are not easy to remove, propolis foraging activity or insufficient surveillance by the beekeeper could be the reason .For all symptoms, it is determined whether they affect a small number of individuals or numerous bees, which would indicate a severe problem. Furthermore, it is relevant which castes of bees show the symptoms.During the colony investigation, the queen is sought. It is observed whether she shows any abnormalities and whether the symptoms are equal to those of the workers. The queen does not always have the same virus load and symptoms as her colony .To investigate the phenotype of bees and pathological changes in more detail and to perform a laboratory analysis, bee samples have to be taken. Most suitable are living symptomatic bees, euthanized. However, in case of increased forager mortality, freshly dead bees from the soil in front of the hive could be appropriate as well, but it has to be considered that there can be false-negative results in virus diagnostics because RNA is unstable in the environment. Different diagnostic methods require a specified number of bees, a fact that should be considered when taking the samples. To assess the size of a sample, the following reference value can be considered: 100 mL are equivalent to approximately 330 honeybees and 31 g. The required number of bees is caught and euthanized by freezing  . Other wVarroa infestation load, are crippled wings in freshly hatched honeybees -Polymerase Chain Reaction (PCR). In the following, the methods are described for CBPV, ABPV and DWV in the way they are performed at the Friedrich-Loeffler-Institute, the Federal Research Institute for Animal Health. A sample of 50 symptomatic bees is an appropriate sample size to submit to the laboratory.TM Dissociator. Total RNA is purified from 150 µL of clarified bee homogenate using the RNeasy Mini Kit  according to the manufacturer’s instructions. For each of the viruses, a one-step real-time RT-PCR is subsequently performed in duplicate using the AgPathIDTM One-Step RT-PCR Kit  in a 96-well reaction plate with 2.5 µL RNA in a final volume of 12.5 µL.From each bee sample, ten bees are homogenized using the gentleMACSFor CBPV detection, it contained 320 nM of forward and reverse primer , and 200 nM of the CBPV probe (CBPV 2 probe: 5′- FAM- TCA AGA ACG AGA CCA CCG CCA AGT TC -BHQ1 -3′) .For ABPV detection, it comprised 800 nM of forward and reverse primer , and 90.4 nM of the ABPV probe (ABPVnTaq: 5′- FAM- ATA GTT AAA ACA GCT TTT CAC ACT GG -BHQ1 -3′) .For DWV-A detection, it contained 350 nM of forward and reverse primer , and 100 nM of the DWV-A probe (Pr-DWV_4293: 5′- FAM- CCT TGA CCA GTA GAC ACA GCA TC -BHQ1 -3′) . For DWVThe thermal cycling conditions are 10 min at 45 °C (RT = reverse transcription), 10 min at 95 °C , followed by 41 amplification cycles at 94 °C for 15 s and 60 °C for 45 s. The results are expressed as the mean of the two replicates for each reaction. Both internal controls monitor that the RNA extraction was successful in all samples (and extraction controls), as well as confirm their transcription into cDNA and the amplification of those during real-time RT-PCR. Furthermore, this allows to see whether a uniform effective RNA extraction took place.Varroa mite is one of the main stressors, probably globally “the greatest threat” [Varroa destructor serves as a mechanical and biological vector for various honeybee viruses and suppresses the immune response of honeybees [The  threat”  for honeoneybees . TherefoTo evaluate the mite load in a colony, different methods are described in the literature. It is possible to examine the debris, the brood and adult bees. Drone brood can be investigated by removing the lids of the cells and studying the larvae and combs individually A. In casVarroa mites are 1.2 to 1.7 mm [Adult bees play a role in assessing the mite load B. ExaminBak et al. compared the flotation and the powdered sugar shake method in 2009 with similar results for both . About 3The second method leaves the bees alive. Dusted with icing sugar, they are shaken gently two times for two minutes. The grooming behavior of the bees is stimulated and the mites’ feet do not stick any longer, thus, the mites are dislodged from the bees . Either Malpighamoeba mellificae and Nosema-species, or be a symptom of stress. In 21 examined honeybee colonies with fecal spots, only 43% could be proven to be Nosema-positive [Fecal spots at or in honeybee hives can be a sign for nosemosis, but they do not have to be . Dysentepositive .Nosema-species can occur in a honeybee colony as a covert infection without harming it. But, it can serve as an additional and weakening stressor to the honeybees. In some cases, it can be a precursor of other diseases by opening infection routes due to epithelial damage in the midgut [e midgut .Nosema and Malpighamoeba mellificae.For investigation of nosemosis, abdomens of 20 freshly dead bees are separated in a mortar A. They aNosema spores are oval-shaped and about 4 to 7 µm in length and 2 to 4 µm in width. The two species, N. apis and N. ceranae, cannot be distinguished by this method [s method .An estimation of the number of spores can be carried out using the scheme described by Ritter in 1996 . The spoIf a massive number of dead bees is found in front of a hive or crippled bees are seen in the colonies, the beekeeper will often suspect a bee poisoning incident with a bee damage as a result of exposure to toxic plant protection products. However, there are some facts which have to be considered. Are all colonies in an apiary affected by increased mortality or is it only one or two? Is the apiary near fields making an application of a pesticide possible? And, are there other symptoms occurring?Nosema-detection is performed, because Nosema-infected bees are more susceptible to intoxications. Finally, a bioassay with larvae of Aedes aegyptii L. is done to detect the presence of bee toxic insecticides. In 2018, 141 samples were sent in with the suspicion of bee intoxication, but only 61 samples were suitable for investigation. In 19 of these 61 incidents, bee toxic insecticides have been detected [In Germany, the Institute for Bee Protection in the Federal Research Centre for Cultivated Plants, Julius-Kühn-Institut (JKI), in cooperation with the national plant protection services examines samples of honeybees with suspected poisoning and plants. The bee sample undergoes a pollen analysis with pollen from the pollen basket (corbicula) or bee hairs to narrow down the incident to plants, which the bees visited. A detected . Furtherdetected .The numbers clearly illustrate the relevance of sending in appropriate bee material. An appropriate sample for this investigation should consist of 1000 bees . The sample should be taken under witness during the first 24 h after seeing symptoms and immediately sent chilled to the investigating institution . The basAfter examinations, the next step is to list medical issues and establish a final diagnosis. According to the list of medical issues, a prognosis is derived, and a treatment plan created. An example in case of an overt CBPV infection can be found in Dittes et al. . InvestiPaenibacillus larvae and Melissococcus plutonius and infestations with Acarapis woodi, Varroa spp., Aethina tumida and Tropilaelaps spp. in Article 1.3.8 [Paenibacillus larvae and infestations with Aethina tumida and Tropilaelaps spp. are notifiable. Currently, virus diseases form a part to the notifiable diseases only in Romania [If a notifiable disease is diagnosed, the relevant authorities have to be informed. There are notifiable diseases to the World Organization for Animal Health (OiE) and to the veterinary authorities in each country, depending on local regulations. The Terrestrial Animal Health Code lists infections with le 1.3.8 . In Germ Romania .Varroa-associated diseases, is to control the Varroa mite infestation. Further studies have to show recommendable therapy measures to control other viral diseases e.g. CBPV in honeybee colonies with overt infections.An infected honeybee colony should be monitored closely during disease management to recognize problems and success as soon as possible and intervene in case of complications. It is advisable to use a logbook to record detailed information on every colony during disease management as well as during the whole season. Veterinarians are familiar with the management and interpretation of patient records as well as drawing conclusions out of them. A detailed logbook is important to gain basic information to choose and apply appropriate therapy concepts for disease control. The main point to prevent DWV infection and disease, as well as further A holistic approach to bee disease diagnosis is important to establish a correct and comprehensive diagnosis and save colonies. Disease outbreaks in honeybees are often tied to more complex interactions than in other species. Especially, the biological form of life in a superorganism has to be considered during the entire course of examination and management. Furthermore, the diagnostic techniques and possible ways of treatment are more limited than in the “normal” veterinary patient. There are no vaccinations and less veterinary medical products available for honeybees. The lifetime of an individual bee and the dependence on the season have to be considered.A detailed and careful investigation is the base of diagnosis, and treatment decisions are to be made focusing on the colony as a whole rather than the individual animal. Depending on the colony location, the number of colonies in an apiary and beekeeping strategies as well as further influencing factors, diagnostics and the management plan have to be adapted to the individual requirements.Veterinarians can be a valuable asset to the beekeeper because of their ability to prevent, diagnose and treat diseases in various species and in populations. In the future, veterinarians should recognize the important role and also the opportunities they can have within the honeybee sector. Taking the chance offered, the veterinarians may be part of education of beekeepers, part of sanitary audits and part of bee health overall in the veterinary practice, in addition to their official function in Veterinary Authorities.