Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. Falling outside these groups are an enormous variety of what appear to be either ssDNA viruses or plasmids that have been recently discovered during metagenomic surveys of terrestrial and aquatic environments [Single stranded (ss) DNA viruses infect animals, plants, fungi and bacteria and are known to cause a variety of diseases in domesticated plants and animals. Their genomes can be linear or circular, multi- or single component and are all smaller than 9 Kb in length . There aronments –7. WhileGeminiviridae, Circoviridae and Nanoviridae, two families of bacterial plasmids and many of the unclassified circular ssDNA molecules directly sequenced from the environment , the liet al. –68.v-ori) [In the circular ssDNA viruses, RCR can only commence following the conversion of ssDNA viral genomes into transcriptionally active covalently closed circular dsDNA molecules by host DNA polymerases (Step 1 in v-ori) ,64,72–77v-ori) ,78). As v-ori) ,77,78. RAlthough it is presently unknown whether the anelloviruses also replicate via a rolling circle mechanism, circular dsDNAs have been detected in the livers and bone-marrow cells of people naturally infected with the anellovirus, Torque teno virus (TTV) ,80. GiveUnlike in circular ssDNA viruses where complementary and virion strand synthesis occur in separate steps, the variants of RHR that occur in linear parvoviral genomes generally involve the replication of both viral strands during the same continuous process . RHR is While the precise details of RCR and RHR mechanisms most likely vary quite substantially between the families , from the perspective of understanding recombination mechanisms at least, the most important and completely conserved feature between these replication mechanisms is that they yield a variety of ds and ssDNA forms that often contain multiple genome copies.2.2.The processes by which recombination occurs within ssDNA viruses are still quite poorly characterized but conceivably involve a number of different mechanisms. For example, interruption of replication by clashes between transcription and replication enzyme complexes or through single stranded breaks in the template strand of replicative form dsDNAs could cause premature detachment of replication complexes. If replication restarts following the reattachment of these complexes to template molecules other than those on which replication had initially started, the resulting fully replicated genomes will be recombinants generated by a mechanism known as copy-choice .Another way in which recombination could potentially occur in ssDNA viruses is via host double stranded break repair pathways. Within cells infected with circular ssDNA viruses undergoing RCR many genomes will occur in the form of either covalently closed or open circular monomeric or multimeric dsDNA molecules  Figure . SimilarStarting from the broken ends of a dsDNA molecule these dsDNA break repair processes probably involve the 5′ to 3′ nuclease digestion (or resection) of the blunt ends to produce 3′ ssDNA overhangs. The 3′ ssDNA ends of broken molecules will then be “matched” up with homologous sequences on unbroken dsDNA molecules (a process known as strand-invasion) following which the 3′ ssDNA ends of the broken molecules will prime the synthesis of a new DNA strands on the unbroken templates .When the template molecule is an unbroken covalently closed circular viral genome, the polymerase complexes producing the recombinationally replicated ssDNA strands can do multiple circuits around the template. These ssDNA strands can therefore potentially grow to three or more times the genome length and are presumably made double stranded by the same host mediated replication processes that convert ss viral DNA into dsDNA ,85.The so-called heterogeneous-length high molecular weight double stranded DNA  moleculWhile it is unknown how common RDR is amongst ssDNA viruses, recombination patterns detectable within viruses isolated from nature display remarkable similarities across many of the rolling circle replicons ,89 whichWhen ssDNA viruses replicated by either RCR or RHR, produce hDNA molecules containing multiple genome copies and multiple replication origins , it is likely that “replicational release” of genome length virion strand molecules will occur from these just as it does from infectious tandemly repeated viral genomes that are frequently used to reconstitute circular ssDNA virus infections from cloned genome components ,90,91. T3.Whereas nanoviruses can have genomes consisting of up to eight circular ssDNAs each ∼1 Kb long ,92–95, gAn important criterion that must be met for genome reassortants to be fully functional, however, is that their reassorted components and the genes they encode must interact efficiently with one another ,110. ForWhereas in begomoviruses the DNA-B and DNA-beta components are trans-replicated by a Rep expressed from the DNA-A component, in nanoviruses it is a “master Rep” expressed from the DNA-R component that trans-replicates the other genome components. The replication origins of the various begomovirus and nanovirus genome components are structurally very similar and reside in a so-called common region that is usually quite highly conserved between the components of a given genome. The common regions of a genome’s various components contain repeated (or iterated) ∼5 nt long Rep specificity determinants  that are specific for the Rep expressed by that genome’s DNA-A (if it is a begomovirus) or DNA-R (if it is a nanovirus) ,111–114.Variation in the specificity with which different Reps interacts with the different genome constituents of multi-component viruses implies that there probably also exist variations in the rates with which different genome components are reassorted in nature. For example, geminivirus beta satellite molecules can be trans-replicated by Reps very distantly related to those of their cognate viruses ,117–119 4.It is probable that homologous recombination plays an important role in the production and adaptation of genome component reassortants. Given that DNA-A will rapidly and efficiently rescue a replication incompetent DNA-B component via homologous recombination ,120,121,Similarly, when a component is introduced into a new genome, part of the capture process can involve replacement of the component’s common region with that of its new DNA-A / DNA-R component ,113,122.It is apparent, however, that simply replacing the common region of a dependent component with that of a new master component will not always produce an optimally functioning multi-component virus. For example, it has been shown in geminiviruses that shortly after a DNA-A captures a new DNA-B, the trans-replication process can be less efficient than that observed between co-evolved DNA-A/DNA-B component pairs . FollowiIn both laboratory experiments and the field, recombination between the geminivirus DNA-A component and its associated satellites occasionally yields satellite molecules that have the DNA-A common region ,123,124.It has been speculated that such recombinants may be a key stage during the formation of multi-component ssDNA virus genomes . It is c5.Genomic rearrangements and deletions frequently arise during anellovirus ,59,133, Sub-full length genome molecules (hereafter referred to as sub-genomics) and genomes with sequence duplications that arise during natural parvoviral infections are possibly the result of DNA secondary structure induced template switching of DNA polymerases at 5–10 nt long direct repeats ,140 duriIn the anelloviruses, there is a tendency for sub-genomics to have breakpoints within either the 5′ half of ORF2 or at GC rich genomic sites . AlthougBy replicating at the expense of full-length genome components it is possible that sub-genomic DNAs that arise during geminivirus infections could reduce symptom severity and, in so doing, act as modulators of viral pathogenicity –149. AltIn geminiviruses, sub-genomics can outnumber full length genomes by as many as ten to one  and whilWhile the significance of sub-genomic molecules in natural geminivirus infections remains unknown, it is likely that the same sequence deletion process that creates sub-genomics results in the rapid reversion to wild-type size of genomic DNA molecules with either insertions or deletions that make them bigger or smaller than full genome length –155. Thev-ori of circovirus genomes and can very rapidly optimize both the rearrangement of disrupted iterated Rep binding site sequences [v-ori hairpin structure [Interestingly, small sequence deletions and rearrangements apparently occur at very high frequencies near the equences  and the tructure . While n6.The recombinational transfer of genetic material between ssDNA viruses and their hosts is known to occur in both directions. Geminivirus sub-genomic and satellite molecules occasionally contain small fragments of non-viral DNA that is presumably host derived ,105. TheNicotiana species: One integration in the ancestral germ line of Nicotiana tabacum, N. tomentosa, N. tomentosiformis and N. kawasamii and another in that of N. tabacum and N. tomentosiformis [Non-homologous recombination between host and viral DNA occasionally results in the integration of ssDNA virus genome fragments into host chromosomes ,160–162.siformis –169.Nicotiana genome have been used as evidence to indicate that distinct geminivirus lineages present in the New and Old Worlds are less likely to have diverged following the breakup of Gondwanaland 100 MYA [Integrated viral genomes are interesting because they provide a view of what ssDNA viral genomes may have looked like millions of years ago and can enable us to determine the timing of important events during the evolution of these viruses. For example, despite extremely rapid short term evolution rates ,24 that  100 MYA  than to  100 MYA .It is also very probable that as more eukaryote genomes are sequenced additional integrated ssDNA virus genome fossils will be uncovered. For example, during our construction of the Rep gene phylogeny presented in 7.The rates of recombination and genome reassortment that occur between different viral species or strains will obviously be strongly influenced by the frequency with which viruses in these different lineages co-replicate within the nuclei of shared host species. Therefore degrees of overlap between (1) geographical ranges, (2) epidemiological cycles, (3) host ranges, and (4) tissue tropisms are all expected to be major determinants of how frequently the individuals within different virus populations recombine with one another. Most well characterized terrestrial ssDNA virus species with non-human hosts seem to have well defined geographical ranges with populations within these species displaying high degrees of spatial structure ,171–176.Epidemiological factors that will determine how frequently viruses that could feasibly recombine actually do recombine will be the numbers of individuals infected with the viruses, the viral titers that are attained within these infected individuals and the durations of their infection cycles. Whereas numbers of infected individuals will determine the frequency with which genetically distinct viruses co-occur within mixed infections, viral titers and infection durations within co-infected individuals will determine the frequency with which the viruses co-occur within the same infected nuclei. For example, it is probable that the large numbers of recombinants observed in many anellovirus species are due to these species having extraordinarily high incidences (often >75%) within their respective host populations ,177,178.Finally, the host range sizes of ssDNA viruses will determine how many opportunities they have to recombine with related species in the environment. A good example of how variation in host ranges can influence opportunities for recombination can be found amongst the geminiviruses of South America. On this continent, an enormous number of geminivirus species have emerged as crop pathogens within the last two decades. This surge in the number of reported geminivirus associated plant diseases has been widely attributed to the introduction onto the continent in the early 1990s of a highly invasive transmission vector strain (or biotype) that feeds on a far wider range of species than indigenous strains –181. By 8.When genetically distinct ssDNA virus genomes co-replicate within the same nucleus there are a number of different mechanistic factors that could determine the patterns of recombination that might occur. Recombination breakpoints that are detectable in ssDNA virus genomes sampled from nature are generally not randomly distributed and have been known to either cluster within discrete recombination hot-spots or to occur much less frequently within recombination cold-spots ,188–190.8.1.v-oris of almost all of these (excluding the nanoviruses) are recombination hot-spots [v-oris are defined by a 10–30 nt long inverted repeat sequence capable of forming a hairpin structure that contains within its loop a highly conserved nonanucleotide sequence that defines the actual v-ori [Full genome sequence analyses of field isolated circular ssDNA viruses that replicate via RCR have indicated that the ot-spots . These val v-ori ,64,191.i.e., by recombination dependent replication [v-ori (see Section 2.2 above).Recombination experiments in geminiviruses  and circlication ), recomb8.2.in vitro recombination experiments [The efficiency with which homologous recombination can be used to replicationally repair broken ss and dsDNA molecules is strongly dependent on the degrees of similarity between broken sites and those of the unbroken molecules used as templates during recombinational repair. Obviously when the broken molecules and their homologous templates are 100% identical this consideration is irrelevant. However, in a mixed infection when the template and broken molecule are not identical, it is expected that recombination will tend to occur most efficiently if the break occurs within a genome region where the broken and template molecules are very similar to one another. This is in fact what is observed during eriments ,194. Cureriments .8.3.v-ori of most circular ssDNA viruses is both a recombination hot-spot and forms a stable hairpin structure and it is conceivable that these two characteristics are not unrelated. In circoviruses, for example, a very credible “melting pot” hypothesis has been proposed to explain how secondary structure in this region facilitates local copy-choice recombination within the stem of the hairpin so as to maintain its palindromic sequence [In many viruses with ssRNA genomes, secondary RNA structures colocalize with recombination hot-spots –200. Thisequence .v-ori hairpin tend to occur at sites where one parental sequence has a predicted secondary structure but the other does not [Computational predictions indicate that additional uncharacterized ssDNA structures probably exist within many ssDNA virus genomes ,201 and does not .It should also be pointed out here that, as we explain later, genomic secondary structures may have another quite different influence on where recombination breakpoints occur within geminivirus genomes. Specifically, it is apparent that whereas the overall genomic secondary structures of recombinant genomes can vary quite substantially from those of their parents, there is evidence of strong selection pressures in geminiviruses for recombinants to maintain parent-like secondary structures .8.4.ssDNA viruses such as the geminiviruses and circoviruses have genes that are expressed from both the virion and complementary sense strands. It is perhaps significant that the complementary sense genes of these viruses are expressed in the opposite direction to RCR because whenever DNA replication proceeds in the opposite direction to gene transcription the opportunity exists for replication complex-transcription complex clashes . Evidenc8.5.Another factor that influences site to site variations in basal recombination rates across ssDNA virus genomes is the association of transcriptionally active viral covalently closed circular DNA forms with host histones and their packaging into mini-chromosomes. Although mini-chromosome formation amongst the ssDNA viruses has only been definitively shown for parvoviruses  and gemiRecombination breakpoint hot-spots detectable within the genomes of geminiviruses in the genus begomovirus co-localize very closely with genome sites that are exposed within mini-chromosomes to host transcription and replication factors ,78,107. 9.As with most other organisms on Earth it is likely that recombination between pairs of nearly identical ssDNA viruses is both a key mechanism in rescuing broken genomes  and an ei.e., when multiple examples of the same recombinant forms have been sampled from the environment they are classified as CRFs). Although the detection of CRFs in many circovirus [It is expected that if such adaptive recombination occurs, the recombinants that it yields should increase in prevalence to the point that they become detectable as circulating recombinant forms  and Tomato yellow leaf curl Sardinia virus (TYLCSV) frequently coexist within mixed tomato infections in these countries and yield a variety of recombinant forms. Although at least some of these recombinants have proven viability ,233,235  Sicily; ,217,233)Furthermore, an interesting feature of the most prevalent and widely distributed of the TYLCVTYLCSV recombinants that have emerged in Spain in recent years is that it has apparently experienced elevated rates of non-synonymous nucleotide substitution relative to its parental virus lineages . As has It is probable that the fitness costs associated with recombination are due at least in part to recombination frequently disrupting co-evolved intra-genome interaction networks . These “10.1.v-ori, the Rep recognition sequences and the N-terminal region of Rep (which encodes the recognition sequence binding site) tend to always be inherited from the same parent. This pattern is consistent with the hypothesis that recombinants with incompatible Rep/Rep binding site sequence pairs will in most cases be replicationally defective [It has been noted in geminivirus recombinants that when parental viruses each have different Rep recognition sequences near the efective . Accordiefective –243.By comparing the viability of pairs of laboratory constructed recombinant viruses with reciprocal gene exchanges to the viability of their non-recombinant parents, it is clear that there are likely numerous such interactions throughout ssDNA virus genomes that must be maintained to ensure the fitness of recombinant genomes ,222,234.The degree of disruption that recombination causes in long-range interactions depends on both the genome regions that are transferred and the degree to which the transferred sequences resemble those that they replace . As a geRegardless of how interactive a transferred genome region is, if the portion of its sequence that is involved in these interactions is identical in both parental viruses such that no interactions are disrupted, then it is likely to continue functioning well following its transfer . TherefoThe constraints that mandatory maintenance of optimal long-range sequence specific interactions place on the viability of recombinants are so severe that patterns of recombination that arise during recombination can actually be used to trace these interactions. Specifically, within the recombinant genomes that arise during a mixed infection of the tomato infecting begomoviruses TYLCV and Tomato leaf curl Comoros virus (TLCCV) there is a significant tendency for genome regions that interact with one another to be inherited from the same parent . This te10.2.Another class of interactions that could potentially be disrupted by recombination are the amino acid interactions that are required for proper protein folding and/or oligomerization. Whereas many amino acids within a protein must specifically interact with one another to ensure proper folding , proteinIn geminiviruses and parvoviruses the impact of recombination induced disruption of protein folding has in fact been directly detectable within the replication associated protein and coat protein genes of recombinants arising both in the field  and duri10.3.In much the same way as recombination between divergent ssDNA viruses can potentially disrupt protein folding when breakpoints occur within protein coding regions, it could potentially disrupt the folding of ssDNA genomes into biologically important secondary structures. Although the importance of genomic secondary structures at the replication origins of many ssDNA viruses is well established –249, it 11.Given the prominence of recombination during the evolution of ssDNA viruses it is reasonable to speculate that their basic genome organizations have most likely evolved to both maximize the adaptive value of recombination and minimize its potentially deleterious effects. For example, the central role of recombination throughout the evolutionary histories of the ssDNA viruses that replicate via RCR is probably reflected in the genomic positioning of their Rep genes and the genome sites that interact with Rep. Generally, replication specificity determinants that interact with Rep and the nucleotide sites encoding the Rep residues that interact with these specificity determinants are within 100 nucleotides of one another and are frequently bounded by recombination hot-spots. This genomic arrangement ensures that, following the transfer of this “replication specificity module” into a foreign genomic background, there is a high probability that it will continue to function properly. As has been indicated in various studies ,224, simWhile the examination of ssDNA virus recombination patterns at the genome scale could provide valuable insights into the genetic architectures of these viruses, use of virus sequence data to estimate recombination rates and patterns of sequence exchange at the population scale could be a powerful means of comparatively studying the epidemiological characteristics of ssDNA virus populations. Specifically, differences between intra-population recombination rates of otherwise very similar virus populations would indicate differences in the rates at which viruses in the populations co-infect individual nuclei. Coupled with incidence and viral load data, recombination rate estimates could be used to differentiate between infection incidences, virus titers within infected individuals, and differences in infection durations as causes for differences in the recombination rates. Such data could prove crucial for modeling epidemiological influences on the recombination patterns seen in ssDNA viruses .While certainly informative, it can also be problematic to directly compare such “population-scaled” recombination rate estimates between different species. It would therefore be very valuable if efforts were made to directly estimate for different ssDNA viruses in the absence of any selection both basal per replication cycle recombination rates and basal genomic site-to-site variations in recombination frequencies. With such data in hand it will be possible to control for mechanistic differences in recombination between species so as to increase the power with which recombination patterns can be used to either identify the intra-genomic sequence interactions that are most strongly preserved by natural selection or infer ecological interactions between species.Since recombining viruses obviously have somewhat overlapping geographical distributions, host ranges, and tissue tropisms, patterns of sequence exchange amongst viruses sampled from nature could also be used to map the ecological interactions between virus species. Whereas the web of genetic exchanges amongst viruses could reveal the virus lineages that are the most promiscuous recombiners (and which are therefore likely to be major contributors to future recombinants), within the context of host species preferences and geographical ranges, such maps could also reveal key environments  and host species  where recombination occurs most frequently. From a purely disease control perspective such information could be extremely valuable when it comes to making policy decisions aimed at reducing the probabilities of dangerous recombinants emerging. Such undesirable recombinants could include those that are more virulent, break inbred/genetically engineered resistance genes, are drug resistant and evade vaccine induced immune responses.As is the case with many RNA viruses, the evolution, dispersal, and population growth/decline rates of ssDNA viruses all occur within similar time-frames such that histories of movements and population size variations of many ssDNA virus species should be detectable using their genomic nucleotide sequences. Besides potential applications of temporally scaled phylogenetic analyses to the dating of important evolutionary events such as host range or vector preference switches, recently developed phylogenetics based approaches could be used to pinpoint the geographical locations where these events occurred ,252,253,


There is a growing interest in beef cattle breeding in Sweden. The majority of the females are bred naturally, which is why it is important to choose healthy fertile bulls to obtain good reproduction and profitability. The breeding soundness evaluation includes measurement of scrotal circumference (SC). Our aim was to analyze if the SC of performance tested beef bulls has changed over the years. In total, 1332 bulls  from 13 batches (1997-2010) were included in the study. Case book entries from final evaluation of the bulls, 11-13 months old, were compiled and analyzed.An overall mean SC of 34.7 cm independent of breed and age was found which is above the set minimum level. Only eleven bulls did not reach the minimum level. An increase in SC of 0.06-0.07 cm/year was shown for all breeds. In all (1997-2010), the increase of the average SC (independent of breed and age) was approximately 1 cm. The positive trend was apparent for all breeds but only statistically significant for the Charolais breed.In conclusion, our results indicated an increase in the SC over time, which improves the possibilities to obtain performance tested beef sires in Sweden with the potential for achieving better fertility results. Bull Breeding Soundness Evaluation (BBSE) procedure, where a general health check, an andrological evaluation, collection of a semen sample and an evaluation of the bull’s mating ability are included [Beef production is currently an increasing agricultural sector in Sweden with more than one third 192 501) of all Swedish cattle being beef breeds 92 501 of. Sub-ferincluded . In the included  have shoincluded ). Severaincluded ). UnfortPreviously, until 2010/11), approximately 170 beef bull calves were tested annually at the performance station in Gismestad near Linköping, Sweden. The bull calves were between five to eight months old at arrival and the testing period was approximately six months. They were selected by each breeding organization based on their BLUP (Best Linear Unbiased Prediction)-values. At the performance testing station the bull calves were gathered in different groups according to breed and were raised together under the same conditions, being evaluated for growth rate, health, sustainability and temperament. Since traditionally there was a focus on their growth rate, the bulls were weighed every second week. In connection with weighing, the testes and epididymides were palpated. If defects or deviations were found at the examination, the bulls were culled. Approximately six months later, when the bulls were between 11 and 13 months old, when only approximately half of the initial number of bulls remained, a final measurement of the scrotal circumference (SC) was done to see if they reached the minimum threshold values set for SC for each breed, and the genital organs were examined for the last time just before the yearly auction . Since the bulls were sold at the auction at a relatively low age (11-13 months) almost half of them were considered not to have a completely normal semen picture yet , and sin/11, apprThe aim of this study was to analyze the change, and possible trend, in the mean scrotal circumference of batches of Swedish performance tested beef bulls at around 365 days of age over time (1997-2010).ad libitum with approximately 60% of the energy provided by silage and 40% by concentrate. The bulls were weighed every second week throughout the testing period (September-March) and at the end of the period, an individual growth index was calculated. Besides growth rate, conformation, temperament and SC were evaluated. The minimum threshold values for SC at 365 days of age used at the performance testing station were for Angus/Charolais 31 cm, Hereford 30 cm and for Simmental 29 cm. Case book entries from the final evaluation occasion of the bulls, before the auction, were compiled and analyzed. The clinical evaluations and scrotal circumference measurements were all made by the same person during the whole study period (1997-2010). First the gait of the bull was assessed at a walking pace and at trot and then the bull was placed in a crush and the testes and epididymides were carefully palpated, the size of the testes assessed, the prepuce inspected and the scrotal circumference measured with a metal scrotal tape .In total, 1332 bulls from four of the most common beef breeds in Sweden  from 13 batches of bulls from 1997 to 2010 were included in the study. All bulls included were housed at the same performance testing station (the only one in Sweden) and on arrival, at approximately six months of age, they were divided into groups of 10-20 animals, based on breed and body weight. Each group was kept in a semi-outdoor pen with a concrete floor and straw as bedding. They were fed a complete-ration feed Data were handled and analyzed using the SAS-program . Mean values, standard deviation (SD) and correlations were calculated, and the variation in scrotal circumference was analyzed using analyses of variance (PROC GLM) according to a statistical model including the effects of breed, and the linear regressions within breed of age, year and weight at evaluation. P-values < 0.05 were considered to be significant.The results showed an overall mean scrotal circumference of 34.7 cm. The mean SC (± SD) for the different breeds and batches are shown in Table Positive correlations were found across breeds between scrotal circumference and age , between scrotal circumference and weight  and between weight and age .For the Charolais breed a statistically significant increase in the scrotal circumference was seen during the studied period  after adjusting for age and weight. For the other breeds  the same trends were seen, in absolute figures, , but the increase was not statistically significant .In total, 11 out of 1332 bulls did not reach the minimum levels for scrotal circumference stipulated at the performance testing station at the time of the final evaluation during 1997-2010.The average scrotal circumferences for the performance tested bulls were 34.7 cm independent of breed and age. The average scrotal circumference, adjusted to 365 days of age for the different breeds, was always above the minimum SC threshold values set for approval at the performance testing station. The averages of the scrotal circumference in our study were, in general, higher than those in earlier studies, complied by Barth . One reaOnly a few bulls, 11 out of 1332 during the studied 13 batches of bulls, were culled at the final evaluation due to too low scrotal circumference. However, the bulls that were culled earlier due to too low scrotal circumference before the final evaluation are not included, which makes the total number of rejected bulls somewhat higher. Since it only concerns very few bulls altogether during the whole time period, according to the veterinarian in charge , the total number of bulls that did not fulfill the minimum threshold values set for the SC at the station was still very low.Our findings concur with previous observations that an increased age results in a higher body weight and also to some extent in a higher scrotal circumference , since aThe results from our study showed, however, that there has been a gradual increase in the scrotal circumference of 0.06-0.07 cm/year for all breeds during the studied period (1997-2010). In total, the increase in the average scrotal circumference (independent of breed and age) among young Swedish performance tested beef bulls was almost 1 cm during these 13 years. Although the positive trends over time were similar for all studied breeds in absolute figures, the increase was only statistically significant for the Charolais breed, which was numerically over-represented compared to the Hereford, Angus and Simmental breeds.Barth  recently made a similar investigation regarding performance tested Angus-bulls in Canada and concluded that there had been an increase in the SC of approximately 1 cm during the last 15 years, which is accordance with our results. This indicates that despite using only the set minimum SC threshold values for selection purposes, a certain increase in the scrotal circumference can be reached over time. In Canada, however, some breeders have taken into account the high heritability of scrotal circumference, and used the average values for the breed in question when making their sire selections instead of the minimum scrotal circumference values, thereby achieving considerable breeding progress by their strategy (Barth 2000).In many countries electro ejaculation is routinely used when conducting BBSE but this technique is prohibited on unanaesthetized animals in Sweden and in many other European countries . CollectOur study showed that the average scrotal circumference measurements for the performance tested beef bulls in Sweden were somewhat higher than those internationally set and that very few bulls did not reach the minimal SC requirements. However, the vast majority of beef bulls used for breeding purposes has not been subjected to any BBSE at all. Thus, information about scrotal circumference measurements is lacking for the majority of the beef bulls used for breeding in Sweden today. This is unfortunate and needs to be more carefully addressed by the breeding organizations in Sweden and also by the insurance companies that, at present, apply no minimum SC threshold values before insuring beef bulls.Our results indicate that it is possible to increase the SC in Swedish performance tested beef bulls over time by applying a minimum threshold value for the scrotal circumference. This trend for an increase in the SC over time may have also improved the possibilities to obtain performance tested beef sires at the auction with increased potential for achieving better fertility results.The authors declare that they have neither financial nor non-financial competing interests.PE participated in the design of the study, collected and analyzed the data, and drafted the manuscript. NL performed the statistical analysis and helped to draft the manuscript. LS participated in the design and coordination of the study, analyzed the data and helped to draft the manuscript. All authors read and approved the final manuscript.


The title and conclusions of the study recently published by Jones et al.  concludepeak viral loads, which was a fleeting condition around the second week after virus exposure. The more important virus levels are those over the long term because of the slow and progressive nature of SHIV. As the authors indicated, and which also is obvious from viewing the charts in Figure 4 from the above study, there was no significant action by DVP in the third through 17th week.With regard to the effectiveness in reducing SHIV virus levels, the only significant effect was in reducing the With regard to effectiveness of DVP in preventing reduction of T helper cells, the data in the study show that, with the exception of one case (monkey CA12), the envelope components alone (DNA and protein) appeared to offer protection that was comparable to the envelope components plus the psoralen- and ultraviolet-inactivated vaccinia virus . The idea that the envelope vehicle without the vaccinia virus might have been as effective as regular live vaccinia virus or DVP was not addressed.p < 0.05 level. Thus, the conclusion that DVP had a protective effect against death is not supported at this time due to absence of statistical significance.With regard to preventing death, the findings do offer weak evidence for the notion that vaccinia virus delivered by any of the three methods  have a protective effect because 66% of the unvaccinated cases died as compared to 6% of the vaccinated. The problem is that the statistical significance of the above difference in protective effect was based on Fisher’s Exact Test. If any cell has fewer than two or three cases, as established by Hanley and Lippman-Hand , there wThe study has much value because of the phenomenal amount of challenging biochemical and immunological work using a wide array of methods which each require expertise, the obvious passion for the development of a potent, readily manufactured vaccine against HIV, the new approach suggested of activating lymphocytes of many types, and other strengths. Nevertheless, due to over interpretation of the data, the main conclusion that DVP has a protective effect, and the implication in the title that the actions of DVP are different from the envelope or vaccinia virus by itself, are not warranted.


Raphanus sativus) in India.Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production. Geminiviridae) have circular single stranded DNA genomes that are encapsidated in twinned quasi-isometric particles and are classified into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus . Intergenic region (IR) sequences (~290 nt) contained conserved nonanucleotide sequence, putative Rep (C1) protein binding sites and the TATA box. The nucleotide identity of full-length genome and IR sequences between RaLCD-associated-Varanasi and RaLCD-associated-Pataudi were 79.6 and 71.6%, respectively, indicating involvement of two distinct species  (~87.7%) Table . PhylogeHowever, RaLCD-associated-Pataudi virus sequence was 95.8% identical to Croton yellow vein mosaic virus-[India] (CYVMV-[IN]) isolates Table  and therThe predicted amino acid sequences of the ORFs of RaLCV-[IN:Var:05] were compared to those of other begomoviruses. TrAP, REn, CP and AV2 of RaLCV-[IN:Var:05] showed maximum amino acid identity  whereas CYVMV-[IN:Rad:Pat:08] and CYVMV-IN iterons sequences were identical (GGGGA-CTC-GGGGGA) (data not shown). The major difference between the RaLCV-[IN:Var:05] and ToLCBDV-[BD:2] was found in spacer sequence of two iterons, as RaLCV-[IN:Var:05] contains AC while ToLCBDV-[BD:2] contains AT. A third iteron was present in 5' end of the intergenic common region (ICR) of RaLCV. Noticeably, besides GC rich region, stem loop region and the TATA Box were conserved for all geminiviruses and all the viruses tested here shared two short sequences, AATGGCA and TAAT, located at the 5'end of the region between TATA box and stem-loop.EF175734) and 1367 nt (GenBank Accession no. FJ593630), respectively. These two betasatellites had structural features similar to those of other betasatellites i.e. a single ORF βC1, satellite conserved region (SCR) and A rich region. The complete nucleotide sequence identity between RaLCV-[IN:Var:05] and CYVMV-[IN:Pat:Rad:08] was 63.2%, indicating that these are distinct betasatellite species. The complete sequence of DNA betasatellite associated with RaLCV-[IN:Var:05] and CYVMV-[IN:Pat:Rad:08] shared maximum nucleotide identity with Tobacco leaf curl betasatellite [Pakistan:Rahim Yar Khan:1998] (TbLCB-[PK:RYK:98]) (83%) and Croton yellow vein mosaic betasatellite [Pakistan:Punjab:2006] (CroYVMB-[PK:Pun:06]) (91.1%), respectively  of betasatellites was cloned from both (Varanasi and Pataudi) RaLCD samples using universal primers . The comORF βC1 of RaLCB-[IN:Var:05] and CYVMB-[IN:Pat:Rad:08] showed maximum nucleotide identity with TbLCB-Pak-NIB 12-1] (89.1%) and CroYVMB-[PK:Pun:06] (93.8%), respectively (Table -1 (89.1%Rep coding region (from nt 2154 to 2519). Bootscan and Simplot analysis also supported these observations  and CYVMV-[IN:Pat:Rad:08] (CRA) genomes were analyzed using two independent methods  and the results indicated definite evidence of recombination within RaLCV-[IN:Var:05] genome Figure . In RaLCs Figure a, b. To To detect possible evidence of recombination in the CYVMV-[IN:Pat:Rad:08] genome, Simplot and RDP analysis were performed with begomoviruse bearing the closest identity viz., CYVMV-[IN], and with two tomato-infecting begomoviruses viz., Tomato leaf curl Gujarat virus-[India:Varanasi:2001] (ToLCGV-[IN:Var:01]) and Tomato leaf curl New Delhi virus-India [India:New Delhi:Severe:1992] (ToLCNDV- [IN:ND:Svr:92]). Simplot and RDP analyses revealed that this is not a recombinant virus; rather it is a derivative of CYVMV-[IN]. Similarly, simplot and RDP analysis carried out for betasatellite molecules DNAs (RaLCB-[IN:Var:05] and CroYVMB-[IN:Pat:Rad:08]) could not reveal any evidence of recombination.N. benthamiana plants by agroinoculation. Radish plants co-inoculated with RLA + RLβ developed systemic symptoms including downward leaf curling within 20 days post inoculation (dpi)  onto radish, tomato and ) Figure , however) Figure . Similar) Figure , while C) Figure ; Table 3) Figure b and 4c N. benthamiana resulted in leaf curling, enations and mild stunting of test plants  or ToLCGV (VB) did not influence symptom severity nor the time required for symptom appearance Figure ; Table 4N. benthamiana plants inoculated with NA + RLβ showed distinguishable symptoms like typical downward leaf curling, interveinal chlorosis and twisting of petiole  can replicate autonomously and move systemically in their natural host (radish), and induce mild leaf curl symptoms. However, in the presence of DNA betasatellite, RLA or CRA induces these symptoms, which appear similar to those observed in the fields in Varanasi and Pataudi, India. Therefore, the RaLCD disease complex is caused by cognate association of DNA-A and DNA betasatellite. Prerequisite association of a begomovirus and satellite DNA-β has also been found to be necessary for typical disease development ,15,34. Heviously ,39. Increviously ,41. Tranthamiana -45. Our thamiana .Croton yellow vein mosaic virus and betasatellite DNA, isolated from RaLCD infected plant could infect tomato, we further extended study to test interaction of CRA and CRβ with predominant tomato-infecting begomoviruses species (ToLCNDV and ToLCGV) from India. Plants (N. benthamiana and tomato) inoculated with mixtures of either combinations of CRA + VA + CRβ, CRA + NA + CRβ, CRA + VA + VB, CRA + NA + NB, CRA + VA + VB + CRβ and CRA + NA + NB + CRβ, produced severe symptoms compared with plants infected with isolates of only one begomovirus species. Viral DNA accumulation (CRA) was also slightly enhanced in plants inoculated with CRA + VA + VB + CRβ or CRA + NA + NB + CRβ inoculated plants compared to plants inoculated with CRA + CRβ. Interestingly, beta satellite accumulation was decreased (upto 60%) in the presence of DNA-B component (either NB or VB) in the above combinations tested. This is in concurrent with earlier observation  and Tomato leaf curl Gujarat virus [ToLCGV]) [Agrobacterium cultures containing respective constructs (Table Trans-complementation with genomic components of tomato-infecting begomoviruses (ToLCGV])  was alsots Table  were mixet al. dCTP by random oligonucleotide-primed synthesis [For southern hybridization analysis, total genomic DNA (8 μg) was separated on agarose gel (1%) and transferred to Hybond-N+ membrane , following standard procedures . Viral Dgment nt 9-506 wasynthesis . Viral DThe authors declare that they have no competing interests.AKS performed the experiments. AKS, BC and SC were involved in data analysis experimental design and writing the manuscript. All the authors read and approved the final manuscript.


Dormant infections can become reactivated years after having been acquired on another continent. Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To determine whether the infections in Europe were acquired from an autochthonous source or associated with travel, we used multilocus sequence typing to compare 100 isolates from Europe  with 191 isolates from around the world. Of the 57 human patient isolates, 47 (83%) were obtained since 1995. Among the 40 patients, 24 (60%) probably acquired the C. gattii infection outside Europe; the remaining 16 (40%) probably acquired the infection within Europe. Human patient isolates from Mediterranean Europe clustered into a distinct genotype with animal and environmental isolates. These results indicate that reactivation of dormant C. gattii infections can occur many years after the infectious agent was acquired elsewhere.Until recently,  Cryptococcus gattii has stepped out of the shadows of its sibling C. neoformans. The latter species mainly infects immunocompromised persons, and C. gattii mainly infects apparently immunocompetent persons. C. neoformans is found globally, and C. gattii has been mostly limited to tropical and subtropical areas in Central Africa, northern Australia, and Central and South America  fingerprint analysis, and several multilocus sequence typing (MLST) approaches .We compared a collection of 107 isolates collected from Europe with 194 isolates collected globally . All isoSTE12a or STE12α locus were performed as described  were amplified by using the preferred primer combinations  , 1× PCR buffer (Bioline), 1.9 mmol/L dNTPs (Bioline), 0.5 U Taq DNA polymerase (Bioline), 5 pmol of both primers  (Amplifications were conducted in a 25-µL PCR mixture containing 37.5 mmol/L MgClerlands) , and ≈10CAP59, the initial amplification primers were used for sequencing reactions. For CAP59, the newly designed forward primer CAP59L-Fwd and the original reverse primer JOHE15438  as described  and JEC21  were used to extract the corresponding sequences for all 10 investigated nuclear loci to serve as an outgroup. The best fitting nucleotide substitution model was determined by using MrModeltest version 2   . Sequence alignments were generated with MEGA version 5 (RH), equal to the Simpson diversity index (D), by using the Microsoft Excel  add-in called Haplotype Analysis , 22 AFLP5/VGIII , 108 AFLP6/VGII , 13 AFLP7/VGIV , and 2 AFLP10/VGIV . From 10 isolates (7 AFLP8 [5 from Europe] and 3 AFLP9 [2 from Europe]), genotypes represented interspecies C. neoformans × C. gattii hybrids. These 10 isolates were excluded from further analysis because amplified fragments of hybrid isolates will result in mixtures of different alleles. The 57 human patient isolates from Europe were obtained from 40 patients  with a haploid genotype is shown in the The 301 patients . The genCAP10 locus showed the lowest overall diversity  and the IGS1 locus showed the highest diversity . The 10-loci MLST dataset showed the highest diversity , followed by the MLST scheme of Meyer et al. (DST = 0.971) (DST of 0.959) (ST = 136 vs. 127).Sequence type diversity was calculated for each of the MLST loci, all 10 loci, and the combined datasets according to Fraser et al. . The CAPC. gattii isolates clustered in 5 monophyletic clusters, which were highly supported and agreed with the AFLP genotypes  were found to be genetically indistinguishable from isolates obtained from animals or the environment in the same area , and all fell within a cluster that could not be linked to an environmental source. The same holds true for the human patient isolates from Greece , which came from the same patient who had no history of travel outside Greece.Several C. gattii AFLP6A/VGIIa, the genotype that caused the Vancouver Island outbreak  in France, which had been obtained from patients who had emigrated from Africa to France and which were genetically indistinguishable from an isolate (IP2001/935–1) from a resident of Senegal  had MLST profiles identical to that of outbreak  (17–19).outbreak . A simil Senegal . This seC. gattii AFLP4/VGI mating-type α isolates from Portugal (IP1997/18) and Belgium (IHEM19725B and –S) was found to be indistinguishable from human patient isolates from the Democratic Republic of the Congo and Rwanda  (C. gattii infection (A set of -type α) . Both phC. gattii AFLP5/VGIII isolate from a 24-year-old immunocompetent patient from Germany (isolate RKI97/310) , obtained from an immunocompetent immigrant from Brazil, which had an identical MLST profile to 6 human patient mating-type α isolates  from Brazil (The single I97/310) . The 2 Co Sweden . Anotherm Brazil .C. gattii isolates from Europe (Infection was acquired outside Europe for 24 of these patients (31 isolates) and within Europe for 16 patients (26 isolates) . Among tC. gattii has been reported as a rare cause of apparently autochthonous cryptococcal infections  of the human patient isolates from Europe were found to have an autochthonous origin in Europe that either could be linked to environmental isolates from the Mediterranean area or that came from patients who had never traveled outside their resident country. A total of 24 (60%) isolates could be linked to C. gattii genotype AFLP4/VGI has also recently been reported from the environment in the Netherlands, but these isolates were not similar to any of the AFLP4/VGI isolates from Mediterranean Europe or to isolate CBS2502, which was isolated in 1957 from a pregnant citizen of the Netherlands, who had never traveled abroad but who died of cryptococcosis . C. C. gattii is emerging in Europe, and the isolates from Europe can be divided into 5 genotypic clusters. Most C. gattii infections in Europe are probably autochthonous, and several infections are proven to have been acquired outside the European continent, e.g., during visits to C. gattii–endemic regions, such as the Vancouver Island outbreak area or before migration to Europe from C. gattii–endemic regions in Africa and South America. Reactivation of dormant C. gattii infections and of infections acquired outside Europe after immune suppression occurs more often than previously assumed. This finding might suggest that C. gattii infections caused by certain genotypes are associated with altered immune status of the human host. Thus, C. gattii is probably a more opportunistic pathogen, as has recently been hypothesized, than a strictly primary pathogen (C. gattii isolates with genotypes AFLP5/VGIII and AFLP7/VGIV were rarely found in Europe and were all acquired outside the European continent. However, these genotypes have frequently been isolated from HIV-infected persons and other immunocompromised patients in Africa and the American continents (C. gattii genotypes and the host’s immune status. Further epidemiologic and immunologic research is needed to unravel this apparent correlation.In conclusion, Cryptococcus gattii isolates, background information of Cryptococcus gattii isolates and GenBank accession numbers for the 10-loci multilocus sequence typing data, primers used for multilocus sequence typing, and sequence type diversity for each of the investigated loci and amplified fragment length polymorphism genotype clusters.Detailed maximum-likelihood phylogenetic analysis based on 10-loci multilocus sequence typing data of


Rabies is a zoonotic disease that, in most human cases, is fatal once clinical signs appear. The disease transmits to humans through an animal bite. Dogs are the main vector of rabies in humans on Flores Island, Indonesia, resulting in about 19 human deaths each year. Currently, rabies control measures on Flores Island include mass vaccination and culling of dogs, laboratory diagnostics of suspected rabid dogs, putting imported dogs in quarantine, and pre- and post-exposure treatment (PET) of humans. The objective of this study was to estimate the costs of the applied rabies control measures on Flores Island.A deterministic economic model was developed to calculate the costs of the rabies control measures and their individual cost components from 2000 to 2011. The inputs for the economic model were obtained from (i) relevant literature, (ii) available data on Flores Island, and (iii) experts such as responsible policy makers and veterinarians involved in rabies control measures in the past. As a result, the total costs of rabies control measures were estimated to be US$1.12 million (range: US$0.60–1.47 million) per year. The costs of culling roaming dogs were the highest portion, about 39 percent of the total costs, followed by PET (35 percent), mass vaccination (24 percent), pre-exposure treatment (1.4 percent), and others (1.3 percent) .This study demonstrates that rabies has a large economic impact on the government and dog owners. Control of rabies by culling dogs is relatively costly for the dog owners in comparison with other measures. Providing PET for humans is an effective way to prevent rabies, but is costly for government and does not provide a permanent solution to rabies in the future. Lyssavirus genus in the Rhabdoviridae family Rabies is a zoonotic viral disease caused by a member of the Control of rabies in dogs is an important means to prevent rabies in humans. Possible control measures include mass vaccination of dogs, culling roaming dogs, quarantining imported dogs, and movement restrictions of dogs. Vaccination of dogs offers a safe and effective means to control rabies as has been reported for some endemic countries Rabies is a costly disease Located in eastern Indonesia, Flores Island is populated by over 1.8 million humans Although there are some economic evaluations of rabies outbreaks published for South and South East Asia An economic model was developed using Microsoft Excel 2010 to evaluate the costs of various rabies control measures and the distribution of the costs among the various stakeholders on Flores Island. The inputs for the economic model were obtained from: (i) relevant scientific literature, (ii) available data on Flores Island, and/or (iii) experts such as responsible policy makers and veterinarians involved in rabies control measures on Flores Island. The values of the input obtained from scientific literature were related to the indicated year of the described study or, if not present, to the year of publication. The cost in different years (http://www.bi.go.id). A sensitivity analysis was performed using add-in software TopRank 6.0 for Excel of Palisade Decision Tools to identify the inputs that were highly influential to the output. Furthermore, the costs of each measure were ranked based on their contribution to the total costs.Where, A deterministic economic model was built to evaluate the total costs of control measures Where, The total costs of rabies control measures in the dog population equal the sum of the costs of six control measures: (i) mass vaccination In the following paragraphs, each control measure in the dog population is explained and detailed economic calculations are given for each, including the inputs.A rabies vaccination program that is free of charge and compulsory for all dog owners has been in effect in the Ende and Manggarai regencies of Flores Island since 2000 The organizational activities include planning the campaign, recruitment and training of temporary vaccinators, and selection activities of the areas. The planning began with a meeting to determine the vaccinators, the budget, and the distribution of campaign information. The vaccinators were veterinary assistants graduated from an animal health and/or a husbandry study program at a university or senior high school. To increase vaccination coverage, a veterinarian occasionally trained local people and community nurses as temporary vaccinators, as in 2008. The Agricultural Department in each regency incurred the available budget for the campaign.The communication activities included development and distribution of materials to inform the local community about the vaccination campaign and to stimulate dog owners to vaccinate their dogs. The campaign information was sent to the heads of the villages, religious leaders, and a radio station, and/or was broadcasted from a car with a loudspeaker once a week before the mass vaccination began. The head of each village was asked to encourage dog owners to bring their dogs to a designated place and/or to confine at home for the vaccinator. Religious leaders were asked to announce the campaign schedule in churches and mosques. The radio station was asked to make announcements on consecutive days before the campaign began. Additionally, leaflets and posters were distributed in public areas.The vaccination activities included the vaccination of dogs and an educational program for the local community. On the day of the mass vaccination, vaccinators, veterinarians, and staff of the Regency Agricultural Department went to rural and urban areas to vaccinate dogs and to educate the local community to keep dogs under supervision. Vaccinations were delivered by subcutaneous administration and required a booster at three months to confer one year’s protection The costs of mass vaccination Where, The vaccination of dogs was administered by a group of temporary vaccinators under close supervision of a veterinarian or public servant. Therefore, costs of vaccinators Where, The number of vaccination days depends on the number of vaccinators who can be supervised by one public servant Where As the vaccination campaign was conducted during working days when the children were at school, we assumed that all the handlers of dogs during the campaign were adult people.The inputs used in the calculations for the costs of mass vaccination in dogs are presented in According to The diagnosis of whether the virus was newly introduced in an area was based on the occurrence of clinical signs in a human who lived in that area, accompanied by test results of suspected dogs in that area. In this case, the regency administrator released a warning regarding the rabies danger, usually followed by mass dog culling in that area. For example, when rabies was introduced to East Flores Regency in 1998 and to Ngada Regency in 2000, each regency administrator decided to cull all dogs throughout the regency Culling any dog freely roaming in a public place, regardless of vaccination status, has been applied in Manggarai Regency . Public places include roads, public parks, traditional markets, and open fields.Culling unvaccinated dogs freely roaming in public places was initiated in Ngada Regency in 2001, and expanded into all other regencies on Flores Island except for Manggarai Regency. This program was not operating well because of a lack of regulation to force people to comply. The culling program was carried out in collaboration between government and local community, and was conducted within villages during the day light by shooting  or by beating the dogs with a stick . The majority of the culling was carried out by the local community and dog owners themselves The cost of culling roaming dogs Depreciation costs of guns and sticks were ignored since these were negligible. The guns were provided by police and army departments and were not special purchased for shooting dogs. The sticks were already available in the village.The costs for an information campaign regarding culling dogs are included as an integral part of the campaign of the mass vaccination program. The inputs used in the calculations for the costs of culling control measures are presented in When a bite from a suspected rapid dog was reported, the veterinary authority (investigators) gathered information from the victim and the dog owner. Officially, the veterinary authority would capture and quarantine the dog for 10 days, but usually the owner or the victim’s family already had killed the dog. In this context, the veterinary authority would collect a brain sample or a head of dog to be sent to the central laboratory in Maros, Sulawesi.Where, Diagnostic testing is an integral part of the control program to obtain accurate incidence data. Therefore, all suspected rabies cases in dogs should be confirmed by clinical samples that are tested at a diagnostic laboratory The total costs of testing suspected rabies dogs depend on the number of samples submitted, transported, and tested and the corresponding cost of the results:Where, The inputs of these calculations are listed in When a brain sample of a suspected dog tested was positive for rabies, the authorities attempted to trace all persons who may have had contact with the dog. Anyone bitten by the dog was vaccinated.The costs for tracing back the human contacts of rabid dogs Approximately, 80 percent of the brain samples tested in the laboratory tested positive for rabies. We assumed that all dogs testing positive The Indonesian government applies a quarantine program of minimal 14-days to prevent reintroduction of rabies through the import of vectors such as dogs, cats, and monkeys to Flores Island.The quarantine costs Where The total costs of rabies control measures in humans equal the sum of the pre-exposure treatment costs Each control measure in humans is explained below and a detailed economic calculation is given, including the inputs.Pre-exposure treatment is effective to prevent rabies in persons who have a high risk of contact with the virus, such as veterinarians, veterinary assistants, laboratory workers and public servants involved in the rabies control program We assumed that there were no opportunity costs for the public servants who received pre-exposure treatment, since expected time needed to provide a vaccination was less than 1 hour per person. The input values of pre-exposure treatment are given in Post-exposure treatment, which is given to persons bitten by a suspected rabid animal, consists of wound cleaning, one dose of immunoglobulin, and four (Zagreb schedule) or five doses (Essen schedule) of vaccine The wound should be cleaned with soap for 15 minutes and antiseptic should be used to reduce the contamination from microorganisms A rabies immunoglobulin injection around the wound is an essential part of the PET because it neutralizes the virus before it invades the nervous system Vaccination of persons bitten by a suspected dog aims to prevent clinical signs of rabies, and delay contributes to post-exposure treatment failure The Indonesian Health Ministry (IHM) We assumed that all people bitten by suspected rabid dogs cleaned their wound with water and soap for 15 minutes in line with the general recommendation.The costs related to rabies immunoglobulin injection Where, The factors associated with the costs of vaccine injection Where, This study not only studied the total societal costs of rabies in Flores Island, it also evaluated the distribution of rabies control costs in terms of private and public costs The costs for dog owners include the loss of the value of dogs due to culling measures and income loss (opportunity costs) due to time lost while bringing dogs to be vaccinated and/or to catch their dogs. For exposed patients, costs include the opportunity costs for the patient and anyone accompanying the patient to get treatment and their transportation costs to a medical center for each treatment. Detailed components of public and private costs are shown in A sensitivity analysis was performed to identify those input parameters  and 4 thTotal costs of rabies control measures during the study period (2000–2011) were estimated to be US$13.40 million, with an average of US$1.12 million (range: US$0.60–1.47 million) per year. The costs of control measures in dogs were about 28 percent higher than in humans. When ranked individually, regardless of control measures in dogs or humans, the costs of culling dogs were the highest, accounting for 39 percent of the total costs, followed by post-exposure treatment (35 percent), mass vaccination (24 percent), pre-exposure treatment (1.4 percent), and others (1.3 percent)   and 7.The total costs of control measures fluctuated during 2000–2006, and tended to decrease in the last five years of the study period . The cosTotal costs of rabies control measures in dogs during the study period were estimated at US$8.58 million, with an average of US$0.72 million (range: US$0.19–1.08 million) per year . CullingThe annual costs of mass vaccination of dogs were approximately US$268,360 , with a mean of US$2.49 per vaccinated dog. The price of vaccine contributed only 18 percent of the total vaccination costs of dogs. Other components were vaccinators, supervisors, meeting and training of temporary vaccinators, the information campaign, capital, and the opportunity costs of dog owners. In addition, the costs of mass vaccination of dogs increased from US$123,760 in 2000 to US$395,760 in 2005, and then fluctuated until 2011. This pattern indicates the government’s performance or commitment to control rabies through mass vaccination of dogs. Because of Indonesia’s autonomy system, the local governments of regencies provide budgets for vaccination control measures in dogs. Therefore, budget decisions regarding vaccination of dogs varied among Flores Island’s eight regencies, and the number of dogs vaccinated in each regency was not the same each year, depending upon budget allocations. Even when the central government (Agriculture Ministry of Indonesia) provides vaccines for dogs, regency budgets for training and hiring temporary vaccinators may determine the final vaccination coverage. This problem might contribute to the declining vaccination coverage in the last three years of the study period (2009–2011). Vaccination costs in 2011 were estimated to be two times lower than those in 2005 , as the Total costs of culling dogs were approximately US$5.2 million, with average annual costs about US$432,460 . The average costs per dog culled was estimated to be US$31.70. A large portion of these costs originated from the lost value of the dogs for the dog owners, which accounted for almost 100 percent of the total costs. Note that the annual costs of culling dogs were highest in the first year of the study period and then tended to fluctuate until reaching their lowest value in 2011 , which wThe annual costs of diagnostic testing of suspected rabid dogs were calculated to be US$ 2,930 . The mean diagnostic costs per sample were estimated to be US$10.50. Interestingly, 53 percent of these costs were for shipping specimens to the rabies diagnostic laboratory and to correspondence of the diagnostic results. Specimens were sent to Maros, Sulawesi because there is no veterinary rabies diagnostic facility on Flores Island.The total costs of rabies control measures in humans were estimated to be US$4.82 million, with the largest portion being the PET costs. The pre-exposure treatment contributed only 3.8 percent of the total costs . These cThe annual costs of the PET were estimated to be US$386,170 , with most of the expenses related with the costs of a series of vaccine injections (99 percent).The costs of PET for the first year of the study period were higher than for the next four years, because of a huge outbreak of rabies and a high number of people being bitten by suspected rabid dogs. The outbreak could be attributed to the higher number of roaming dogs. In 2001, the number of dogs decreased as a result of the culling control measure in 2000. The PET costs tended to increase, starting in 2001  until 2010  . The totOf the total costs of rabies control measures, public costs were higher (US$6.8 million) than private costs (US$6.6 million). The majority of public costs (71 percent) were incurred by the Public Health Department, which provided human vaccine and immunoglobulin for free to the local community. In addition, the annual proportion of public costs allocated by the government increased over time, with exception of 2000 . This inThe total costs of rabies control measures were most sensitive to the dog value. An increase or decrease of the dog value by 10 percent resulted in a 4 percent change in total costs. Other input parameters that influenced the total costs in our analysis were the price of human rabies vaccine and the number of vaccine doses in humans; a 10 percent increase or decrease in these parameters, resulted in both cases in a 2 percent change in total costs. Other inputs contributed to changes in the default total costs of less than 2 percent.A deterministic economic model was developed to evaluate the costs of rabies control on Flores Island during 2000–2011. With this model, we calculated the total costs of rabies control measures as they were carried out on Flores Island, by integrating available epidemiological and economic data, scientific literature, and information from experts in rabies control measures. The results are an estimation because some inputs  were uncertain in the analysis. The described analysis is an ex-post analysis. However, the developed calculation model is set up in such a way that it can be used to predict the costs of future rabies control programs , not only for Flores Island but also for other regions or countries.Some limitations of this study may have led to over- or under-estimation of the total costs of control measures. For example, the costs of control measures in humans might have been overestimated because we assumed all people were injected with four doses, despite the fact that the dog-bite patients might receive fewer than four doses in reality. Moreover, the epidemiological surveillance and research costs were not considered in the analysis because of a lack of data. Also, the costs for diseased livestock and human patient cases were not included, which may have led to an under-estimation of the costs of rabies. In none of the regencies in Flores cases of rabies in livestock have been reported, although the Husbandry Department of East Nusa Tenggara province provides the livestock owners a format to report any rabies cases in the livestock. So this omission is, most probably not related with a large under-estimation of the costs of rabies. However, no data were available on the number of patients that were hospitalized due to rabies. Unfortunately, therefore, we were not able to make an estimation of the costs for human patients. Despite these limitations, the estimate made in this study illustrates the economic burden of rabies control measures for all stakeholders on Flores Island, Indonesia as realistic as possible. Our results show that the costs of culling roaming dogs were the highest portion (39 percent) of the total costs, with average costs per dog culled at US$31.70. This finding contrasts with other studies that found the highest portion of costs were for PET The second largest costs for rabies control measures were those of PET, an average of US$178 per patient. The expensive human rabies vaccine and/or immunoglobulin This study also found that the annual costs of PET increased in the last seven years of the study period, which reflects the increased number of dog-bite patients who received PET  as the vThe current control measures in the dog populations were not successful in reducing the number of human bite cases by suspected rabid dogs and rabies as such is still endemic in Flores Island. Some explanations that may contribute to this situation; (1) there was no island-wide dog vaccination campaign as, for instance, carried out on Bali Island Furthermore, of the total dogs registered, the percentage of vaccinated dogs was less than 100%. There are two possible reasons that could explain this situation as described in detail by The culling program of dogs in infected areas failed to prevent the virus spreading throughout the island since not all local people were willing to participate in culling dogs. Only a few local people (approximately 5–10 people in each village) joined as volunteers in the culling of dog procedure.The annual total costs of control measures in humans increased over the years, a finding that contrasts with other studies in different countries. In many countries, rabies control measures in dogs have substantially reduced the costs of PET in humans because fewer people seek PET This is the first study to consider the value of culled dogs in rabies control. This factor was included because dogs have an economic value and are culturally very important for the local society Therefore, culling as a control measure might be less acceptable for a local community because of ethical, social, and economic reasons. In this context, the World Organization for Animal Health Our results demonstrate that the rabies control measures were costly to society. Optimization of the current control measures could reduce the economic burden of rabies in the future. An economic study that weighed the trade-off between controlling rabies in dogs and PET in humans is needed to determine if more control among dogs would be cost beneficial. This study’s results could provide baseline data for additional effectiveness studies.This study shows a generic and transparent way to calculate the societal costs of rabies in a certain region. Rabies has a large economic impact on government and the dog owners of Flores Island. Control of rabies by culling dogs is relatively costly for the dog owners in comparison with other control measures. Providing PET is an effective way to prevent rabies casualties in humans, but is costly for the government, without providing a permanent solution for rabies control in the future. The developed model can be used for future economic ex-ante and ex-post analyses on rabies control.


Revisiting an original idea by Hollerbach , previou A persistent and puzzling issue in motor control is how different motor elemental pieces may be put together into a single unit of behavior. Creating new forms of behavior by combining already existing units is a critical and amazing adaptive ability of living beings on various time scales. Regarding motor behavior, many studies have been devoted to the issue  development during ontogeny eventually involves putting elemental pieces of behavior together and/or taking them apart, a good deal of work has addressed these issues from a developmental perspective. At a theoretical level, a unique framework is Piagetian theory  volunteered for the experiment. Hand dominance was determined by a questionnaire of hand preference in various daily tasks  aged 25–42  of the performed trajectories were digitized at 100 Hz, fed back on-line for display on top of the current model shape on the tablet, and stored for later processing.All shapes, inscribed within a circle of 2 cm of diameter, were displayed at the center of a 21 × 15 cm backlit digitizing tablet (Wacom PL300), which was inserted in a table of adjustable height facing the participants. As soon as the stylus touched the tablet, the sw, the time needed to change pattern. Specifically, Figure x and y components of the traced trajectory. Panel C presents the continuous relative phase in degrees between those oscillatory components calculated through a Hilbert transform routine  behaves over the entire procedure, especially in terms of the stability of their oscillations as a function of the three periods of the experimental procedure . Second, we consider their collective behavior, namely, the stability of the relative phase between the oscillations of the components. Finally, we examine the properties of the switching between pairs of shapes, in order to test our main hypotheses.Results will be presented in three subsections. First we analyze how each individual component  and over the four required relative phases  for each set of shapes separately .In order to assess the stability of oscillation, we used the F = 79.04; p < 0.05 and F = 40.51; p < 0.05, for the diagonal shapes and the cardinal shapes respectively. For all patterns and transitions, the variability of the component period of oscillation was significantly lower for the pre-switching than the post-switching period. However, results did not reveal any difference in variability of the oscillation period as a function of the pattern produced. This finding rules out the possibility that variations in the stability of the relative phase between the x and y components due to their coupling as a function of the produced relative phase be a mere result of variations in the periodic behavior of the individual oscillators.For each set, an Oscillator (2) × Pattern (4) × Period (2) ANOVA with repeated measures on all factors revealed a main effect of Period, We compared the accuracy and the variability of the produced relative phase between the pre- and post-switching periods  and over the four required relative phases  for each set of shapes separately .F = 48.46, p < 0.0001, and the cardinal shapes, F = 57.61, p < 0.0001. Figures Post-hoc Newman-Keuls tests indicated that the 0° pattern was the most stable, while the 180° pattern was more stable than 60°, which in turn was more stable than 120, all ps < 0.01. This is important, because the relative stability of the patterns determines the switching time needed to go from one shape to another. Note that the error in producing the required relative phases followed the same order: The most stable patterns were also the most accurate, a finding already reported in our previous studies on bimanual coordination  × Period (2) ANOVAs with repeated measures on both factors were performed on the AE and the SD and of the relative phase for the two sets of shapes. No effect turned out to be significant for the AE of relative phase in any set. In contrast, the ANOVA revealed a significant Pattern effect for the diagonal shapes, sw, as a function of the Direction of switching for the diagonal (Panel A) and the cardinal shapes (Panel B). Results revealed that irrespective of the patterns involved in the transition, switching from a less stable to a more stable pattern (Direction 2) was significantly shorter than the other way round (Direction 1). The mean difference was 97 ms, F = 13.82, p < 0.01, for the diagonal shapes, and 35 ms, F = 5.03, p < 0.01, for the cardinal shapes. Another finding was that switching between cardinal shapes . A Pair (6) ANOVA with repeated measures reveals a significant effect, F = 10.37, p < 0.0001. A post-hoc analysis showed that the switching time for 0°/60° and 120°/180° transitions (358 ± 47 ms) were similar and significantly shorter than for 0°/180°, 0°/120°, 60°/180°, and 60°/120° transitions (522 ± 50 ms). Moreover, switching times for 0°/180°; 0°/120°, 60°/180°, and 60°/120° did not differ, with a single exception for 60°/120° that was significantly shorter than 0°/120°. These findings indicate that switching between diagonal shapes that were in the same orientation but with different eccentricities  was faster than for shapes that required a change in orientation.Figure sw, as a function of the pairs of cardinal shapes involved in the transitions, D1 and D2 pooled. A Pair (6) ANOVA with repeated measures reveals significant effect of Pair, F = 5.17, p < 0.001. By and large, Figure Post-hoc analyses confirm that transition between 0°/60° and 120°/180° (327 ± 36 ms) were faster than the others (472 ± 42 ms), with transitions between 60°/120° differing from those between 0° and 180°. Here again, switching between cardinal shapes that were in the same orientation but with different eccentricities was faster than for shapes that required a change in orientation.Figure sw as a function of the absolute difference between the starting and final relative phases composing all the pairs of shapes. In such a display, τsw grows linearly with the difference in relative phase . Whether due to a change in eccentricity or in orientation or both between the two required shapes, the larger the change in relative phase is, the longer it takes to perform the switching.The latter effect, valid for both orientations, can be further exposed by analyzing the switching time necessitated to perform a given change in relative phase required by the task, irrespective of the actual shapes to be produced, namely, its shape and orientation. Figure In line with a dynamic approach to coordination, the present study confirms that stability is a theoretical concept and an empirical property of coordinated behavior. In particular, stability is crucial to understanding the production of fairly simple graphic shapes, namely ellipsoids, which are conceived of as basic primitives for real handwriting  are always shorter than transitions between shapes of different orientations. Previous work on 2D trajectory formation  periodically moving about joints. Such independence of coordination dynamics from the effectors, a property already reported in Kelso and Zanone . Yet, producing the same graphic shapes in various orientations implies an altogether different mobilization of the joints and muscles of the hand and the wrist. Simply put, it involves radically different motor commands. Therefore, in a dynamic framework, the two orthogonal coupled oscillators constitute an abstract model “living” in the space of relative phase—and probably somewhere in the brain, as an outcome of the collective, synchronized activity of the neurons—as opposed to a more physical or applied model d Zanone , may be d Zanone , that isd Zanone . As wellSecond, handwriting speed is not only dependent on how many changes in relative phase are required for producing a given trace, each demanding a given amount of time, but also on how big such jumps are. The tight relationship reported here Figure  between A third general lesson is that knowing how transitions between graphic units occur provides a key for understanding coarticulation, a phenomenon typical of speech  variable that carries information about where the trajectory being traced is heading next: A slight modulation about a stable relative phase might be a clue for a forthcoming switch to another relative phase. Indeed, the visual system proves to be pretty sensitive to variation in relative phase in various dynamic displays (Zaal et al., Finally, the foregoing arguments convey an implicit conjecture that producing and perceiving relative phase coordination patterns and their outcome on the paper share common processes, or dynamics as we would say. There is growing evidence, gathered first in the field of speech science in the fifties (e.g., Liberman and Mattingly, In the same line of thought, one has to keep in mind that handwriting is a complex skill that goes beyond the coordination dynamics explored here, which accounts for the lawfulness of the generation process of the graphic trace. For sure, the establishment of any efficient writing system must have implied a coevolution with the corresponding reading system. Thus, the preferred motor output determined by underlying coordination dynamics has been co-defined by the perceptual categories that discriminate between the visible traces to form a coherent and meaningful perceptual pattern. This has certainly been a long process through mankind's history (e.g., Changizi and Shimojo, 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.


Serum samples were obtained from 252 horses in the State of Yucatan, Mexico, from July to October 2002. Antibodies to West Nile virus were detected by epitope-blocking enzyme-linked immunosorbent assays in three (1.2%) horses and confirmed by plaque reduction neutralization test. We report the first West Nile virus activity in the State of Yucatan. Flavivirus, family Flaviviridae (West Nile virus (WNV) is a member of the Japanese encephalitis virus complex within the genus In response to the incursion and rapid spread of WNV in North America, we established equine and avian infection surveillance in Yucatan State, Mexico, in March 2000. Yucatan State is a likely point of incursion of this virus into Latin America because this area is a principal landfall for many species of birds that migrate from the northeastern and midwestern United States  assays and epitope-blocking enzyme-linked immunosorbent assays (ELISAs) at the Universidad Autonoma de Yucatan in Merida. HI assays were performed by using Saint Louis encephalitis virus (SLEV) antigen as previously described (Six horses had evidence of flavivirus infection by HI assay or ELISA . Serum s>90% reduction in the number of plaques (PRNT90).Serum samples positive for flavivirus antibodies by HI assay or ELISA were tested by plaque reduction neutralization assay (PRNT) to identify the infecting virus. PRNTs were conducted in the BSL-3 facilities at Colorado State University. Serum sample results shown to be negative by HI assay and ELISA were not tested. PRNTs were done by using WNV (strain NY99-35261-11), SLEV (strain TBH-28), Ilhéus virus , and Bussuquara virus . Virus stocks were obtained from the World Health Organization Center for Arbovirus Reference and Research, maintained at the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO. We tested serum samples for neutralizing antibodies to SLEV because the virus is enzootic in the Americas and because antibodies to WNV and SLEV often cross-react. Furthermore, horses are susceptible to natural SLEV infections, although clinical manifestations have not been reported . We plan to isolate and amplify viral sequences from migratory and resident birds, as well as from specimens from other seropositive animals, to determine the origin of the WNV strain in Yucatan State. We also provide serologic evidence for WNV infection in horses in Coahuila State (