Dairy-origin beef accounts for a considerable proportion of the prime beef supply. Yet, the beef production potential of dairy bred calves is declining following years of single trait selection for attributes such as milk yield. Producing these animals as bulls allows for increased growth rates and efficiency in comparison to steers, however these systems often involve a high level of concentrate feeding. Thus, creating a system that is subject to fluctuating feed prices and beef market volatility. This objective of this study was to evaluate four production systems, which differed in terms of diet during the grower period. The impacts on health, performance, carcass characteristics, and meat quality were all considered. Our research shows that ad libitum concentrate feeding throughout the production system resulted in superior carcass weights. Increasing the proportion of grazed grass in the diet during the grower period, did result in a reduced carcass weight in comparison to ad libitum concentrates, however total concentrate intake, and therefore the production costs during the grower period were also reduced. Production system had no effect on health or meat quality. In conclusion, including a gazing period within bull beef production systems may be a more sustainable approach to producing Holstein bulls.p < 0.001) and total finishing DMI (p < 0.001) differed between production systems for both AB and SB bulls, with that of GA bulls being the greatest in both cases. Average daily gain (ADG) during the grower period was greatest (p < 0.001) for the HA production system in the AB bulls and the GA and HA production systems for the SB bulls. However, during the finishing period, G bulls had the greatest (p < 0.001) ADG of the AB bulls, while that of the SB bulls was from the G2 production system (p < 0.001). For both AB and SB, bulls on the GA and HA production systems produced heavier cold carcass weights than the G and G2 bulls (p < 0.001). There was no significant difference (p > 0.05) in health, carcass conformation, fat classification, or meat quality between production systems.The aim of this study was to evaluate the effect of production system on the health, performance, carcass characteristics, and meat quality of autumn born (AB) and spring born (SB) Holstein bulls. The study involved a total of 224 Holstein bulls and was conducted over two years . The four production system treatments differed during the grower period and consisted of: (i) grazed with no concentrate supplementation (G), (ii) grazed with 2 kg concentrate supplementation per day (G2), (iii) grazed with ad libitum access to concentrates (GA) and (iv) housed with ad libitum access to concentrates and grass silage (HA). All bulls were finished on ad libitum concentrates and grass silage and were slaughtered at a mean age of 15.5 months. Total grower dry matter intake (DMI) ( The UK dairy herd is predominantly of the Holstein-Friesian (HF) breed, with a major emphasis placed on maximising milk yield . As a reThe ability for bulls to out-perform steers in terms of growth rate and feed efficiency has been widely documented within the literature. Steen  reportedGrazed grass is the cheapest form of feed for ruminants , and thuThe objective of this study was to compare the health and performance of Holstein bulls on four differing production systems to identify if a grazing period could be included in Holstein bull beef production, and if concentrate supplementation at pasture was required. Specifically, we examine the hypothesis that performance will increase linearly with increasing concentrate supplementation at pasture, and that these differences in live weight will still be evident at slaughter. In addition, we hypothesise that pneumonia incidence will be greater at the beginning of the finishing period, for those bulls which have been grazed during the summer. Finally, due to a homogenous finishing period, meat quality will be unaffected by production system.This study was undertaken from May 2017 until June 2019 at the Agri-Food and Biosciences Institute (AFBI), Hillsborough, Northern Ireland. All experimental procedures used in this study were conducted in compliance with the United Kingdom Animals (Scientific Procedures) Act 1986.n = 112) and 2018/19 (n = 112)). In each year a group of 56 autumn born (AB) and a group 56 spring born (SB) bull calves were selected. These bulls were assigned to one of four production system treatments, which differed during the grower period and had a homogenous finishing period, the durations of which are reported in This study involved of total of 224 Holstein bulls and was conducted over two years .G, G2, and GA were rotationally grazed in a 4 paddock system. A 7.2 ha mature perennial ryegrass pasture was divided into four equal blocks of 1.8 ha, with each block subdivided into a further six paddocks, three paddocks of 0.38 ha for AB bulls and three of 0.21 ha for SB bulls. Divisions between paddocks consisted of two strands of electric wire. Each of the six treatment groups at pasture were assigned one paddock per block. All cattle were grazed in one block at a time, and therefore were all rotated on the same day. Cattle were moved when one group (primarily G or G2) reached a post-grazing height of approximately 6.5 cm. A group of 30 spare cattle were then used to graze out the remaining paddocks (particularly GA) in order to bring all paddocks down to the same post-grazing cover. No live weight or intake data was recorded for the spare cattle.Post turnout, G2 and GA were offered concentrates in a trough, G2 were fed 2 kg concentrates once daily throughout the trial. GA were gradually built up to ad libitum concentrate feeding, at the same rate and frequency as HA, at which point a creep feeder was introduced. The duration of the grower period is shown in All bulls were housed on slatted accommodation during the finishing period, the mean duration of which is shown in All bulls were weighed on two consecutive days prior to the commencement of this trial, at the end of the grower phase and at slaughter. The mean of the two weights was taken to be the animals’ LW on each of the three occasions. Throughout the trial bulls were weighed fortnightly to monitor performance. As two of the production system treatments included ad libitum concentrate feeding, bulls were always weighed full, at approximately the same time on each occasion. All weighbridges were calibrated to ISO9001 standards. Average daily gain (ADG) for each phase was determined by the difference between the initial LW and final LW, divided by duration (days) of each phase. Bulls were monitored daily for clinical signs of ill health. The incidence of pneumonia and lameness were recorded  together with any antibiotic treatments given. Rumen temperature boluses  were administered at the beginning of this trial to 203 bulls. Temperature alerts from the boluses were used as an additional tool to assess health status.Pre- and post-grazing heights were measured using a Jenquip rising plate meter (model EC09)  and the difference used to estimate intake. Group intakes of silage and concentrates were recorded daily and refused feed was removed and weighed twice weekly prior to fresh feed being offered. A 10% fresh weight refusal rate was considered acceptable for ad libitum intakes. Mean estimated individual intakes were determined by dividing the group intake by the number of animals per group.Pre- and post-grazing grass samples were analysed weekly using NIRS for crude protein (CP), acid detergent fibre (ADF), water soluble sugars (WSS), and metabolisable energy (ME) using the methods described by Park et al.  and MorrBulls were transported 42 km in a lorry to a commercial abattoir on the morning of slaughter. Bulls remained in one group during lairage and were then slaughtered using the commercial abattoir procedure . At slauA post-mortem examination on the lungs, liver, and heart was conducted following slaughter by the veterinary inspector. Lungs were inspected for signs of pleurisy and pneumonia, the liver for abscesses and peritonitis, and the heart for pericarditis. Each organ was scored on a scale of 1 to 4 .longissimus dorsi muscle was scored for marbling using the Meat Standards Australia scoring system [longissimus dorsi muscle on both sides of the carcass, the mean of these values was taken as the subcutaneous fat depth for each animal [At 24 h post-slaughter, carcasses were split between the 9th and 10th rib, and the g system . Subcutah animal ; the mealongissimus dorsi muscle were removed from the anterior fore-rib joint at three days post-slaughter [ult) and colour (L*(lightness), a*(redness) and b*(yellowness)) were assessed using a Jenway 370 pH meter and a Chroma Meter CR-400, respectively. Both instruments were calibrated prior to measurements being taken. The illuminant for the Chroma Meter was D65, while the angle of observation was 2°. D7 and D14 samples were then vacuum-packed and frozen, where they were stored until further measurements were taken.Two samples from the laughter . These mSamples were left to thaw over a period of 24 h at 4 °C. Following this, they were removed from the packaging and left to bloom for 40 min. Samples were vacuum-packed and cooked in a water bath at 70 °C for 50 min. Samples were weighed pre- and post-cooking, and cooking loss was calculated as follows:Warner Bratzler Shear Force (WBSF) measurements were completed on each sample using an Instron 3366 Universal Testing Instrument. Samples were cored parallel to the longitudinal orientation of the muscle fibres and ten cores (sub-samples) were taken from each meat sample. All sub-samples were of the same diameter (12.5 mm), and were sheared perpendicular to the muscle fibres. The mean maximum load of the 10 sub-samples was considered as the WBSF value for each meat sample.All statistical analysis was completed using Genstat (19th edition). Summary statistics were conducted on the composition of feed. The AB and SB bulls were analysed separately due to the differences in the durations of the grower and finishing periods outlined in Health variables (pneumonia incidence and lameness incidence) were modelled using generalised linear mixed model methodology with production system as a fixed effect. The random effects differed according to which period the health variables related to; for the grower period, year was used as a random, the finishing period consisted of year and pen number, while the slaughter health variables included year and slaughter date as random effects. The binary variables used a binomial distribution with a logit link function, while the count variables used a Poisson distribution with a logarithmic link function.p < 0.001). The greatest concentrate DMI (p < 0.001) and total DMI (p < 0.001) were observed for the GA production system. The AB bulls saw the greatest ADG and housed weight for the HA production system (p < 0.001) (p < 0.001) housed weight and lowest ADG (p < 0.001)  . For the< 0.001) .p < 0.001) between production systems during the finishing period, for both AB and SB bulls (p < 0.001), resulting in a slaughter live weight of 34.1 kg lighter than the GA bulls (p < 0.05). While the SB bulls saw a 68.5 kg difference between the G and GA bulls (p < 0.001).Forage, concentrate, and total DMI differed significantly (SB bulls . The AB p < 0.001) in concentrate usage can be achieved of up to 650 kg for AB bulls and up to 890 kg for SB bulls between the G and GA production systems. p < 0.05) and SB bulls (p < 0.001). In the case of the SB production systems, the lowest ADG was observed from the G production system, while that of GA and HA were the greatest.Total concentrate DMI  shows thp < 0.001) carcass hot weight and cold weight than G and G2 (p < 0.05) between production systems for AB bulls, together with the weight of internal fat (p < 0.001). The remaining carcass traits and meat quality parameters were not significantly different (p > 0.05).For the AB bulls those on the GA and HA production system had a greater (G and G2 . Carcassp < 0.001) lighter than the other three production systems (p < 0.001). Carcass gain for this production system also proved to be the lowest of the four SB production systems. Again there was no significant difference (p > 0.05) between the remaining carcass traits or meat quality parameters.Similar results were obtained for the SB bulls, with the cold carcass weight of the G bulls being significantly (p > 0.05) in the incidence of pneumonia or lameness during either the grower or finisher period for both AB and SB bulls. Furthermore, a number of health variables from the grower and finisher period were so insignificant that they could not be analysed. In addition, no significant (p > 0.05) difference in the incidence of liver abscesses, pericarditis, or pneumonia were identified during a post-mortem examination of AB and SB bulls.The results for the health variables are not presented as there was no significant difference  saw a considerable increase in ADG during the finishing period. This was somewhat greater than that achieved by the HA bulls, and that which has previously been reported within the literature ,34. ThesKirkland et al.  reportedThe SB bulls on G and G2 production systems reached similar slaughter and carcass weights as those slaughtered at 16 months on a 50:50 silage to concentrate diet by Kirkland et al. .Although there was no difference in carcass fat classification or subcutaneous fat depth, differences in internal fat were observed for AB bulls. This contrasts with the results of Therkildsen et al.  who founHealth status and pneumonia incidence were not impacted by production system in this study which is in agreement with previous research . FurtherIn conclusion, the results of this study demonstrate that both AB and SB bulls on ad libitum concentrates (GA and HA) had superior performance during the grower period achieving ADGs of up to 1.58 kg/d. In contrast, AB G and SB G2 bulls had elevated growth rates during the finishing period , and likely exhibited a degree of compensatory growth during this time. However, differences in cold carcass weight were still revealed, with that of G and G2 being lighter than GA and HA, for both AB and SB bulls. Nevertheless, G and G2 production systems did result in a lower total concentrate intake than GA and HA, which would assist in reducing production costs. Instrumental meat quality and animal health were unaffected in this study.


The antiviral protein kinase R (PKR) is an important host restriction factor, which poxviruses must overcome to productively infect host cells. To inhibit PKR, many poxviruses encode a pseudosubstrate mimic of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), designated K3 in vaccinia virus. Although the interaction between PKR and eIF2α is highly conserved, some K3 orthologs from host-restricted poxviruses were previously shown to inhibit PKR in a species-specific manner. To better define this host range function, we compared the sensitivity of PKR from 17 mammals to inhibition by K3 orthologs from closely related orthopoxviruses, a genus with a generally broader host range. The K3 orthologs showed species-specific inhibition of PKR and exhibited three distinct inhibition profiles. In some cases, PKR from closely related species showed dramatic differences in their sensitivity to K3 orthologs. Vaccinia virus expressing the camelpox virus K3 ortholog replicated more than three orders of magnitude better in human and sheep cells than a virus expressing vaccinia virus K3, but both viruses replicated comparably well in cow cells. Strikingly, in site-directed mutagenesis experiments between the variola virus and camelpox virus K3 orthologs, we found that different amino acid combinations were necessary to mediate improved or diminished inhibition of PKR derived from different host species. Because there is likely a limited number of possible variations in PKR that affect K3-interactions but still maintain PKR/eIF2α interactions, it is possible that by chance PKR from some potential new hosts may be susceptible to K3-mediated inhibition from a virus it has never previously encountered. We conclude that neither the sensitivity of host proteins to virus inhibition nor the effectiveness of viral immune antagonists can be inferred from their phylogenetic relatedness but must be experimentally determined. Most virus families are composed of large numbers of virus species. However, in general, only a few prototypic viruses are experimentally studied in-depth, and it is often assumed that the obtained results are representative of other viruses in the same family. In order to test this assumption, we compared the sensitivity of the antiviral protein kinase PKR from various mammals to inhibition by multiple orthologs of K3, a PKR inhibitor expressed by several closely related orthopoxviruses. We found strong differences in PKR inhibition by the K3 orthologs, demonstrating that sensitivity to a specific inhibitor was not indicative of broad sensitivity to orthologs of these inhibitors from closely related viruses. We also show that PKR from even closely related species displayed markedly different sensitivities to these poxvirus inhibitors. Furthermore, we identified amino acid residues in these K3 orthologs that are critical for enhanced or decreased PKR inhibition and found that distinct amino acid combinations affected PKRs from various species differently. Our study shows that even closely related inhibitors of an antiviral protein can vary dramatically in their inhibitory potential, and cautions that results from host-virus interaction studies of a prototypic virus genus member cannot necessarily be extrapolated to other viruses in the same genus without experimental verification. The ongoing SARS-CoV-2 pandemic underscores the urgent need to understand barriers to cross-species transmission of viruses. While the world’s focus is currently on coronaviruses, poxviruses have a history as one of the most deadly human pathogens in the form of variola virus (VARV), and the continued emergence of monkeypox virus (MPXV) and cowpox viruses (CPXVs) suggest that poxviruses continue to represent immediate public health threats. Poxviruses are a large family of double-stranded (ds) DNA viruses encompassing two subfamilies and 14 currently recognized genera, which include many viruses of both medical and veterinary importance. Poxviruses, as a group, infect a large range of animals; however, the host range of individual genera and virus species can vary dramatically [Members of the well-studied orthopoxvirus genus have one of the broadest host ranges of all poxviruses with vaccinia virus (VACV), CPXVs and MPXV infecting a wide range of mammals. Yet the genus also includes viruses that only infect a single species such as VARV, the causative agent of smallpox in humans, and ectromelia virus (ECTV), which only infects mice. There are also orthopoxviruses with a narrow host range such as camelpox virus (CMLV), which primarily infects camels but can also infect humans ,3. This One important host antiviral protein that is targeted by poxvirus host range factors is protein kinase R (PKR), which exerts its antiviral effect against many virus families. PKR is ubiquitously expressed in host cells in an inactive monomeric form. In the presence of dsRNA, produced during the replication cycle of most viruses, PKR is activated through a process of dimerization and autophosphorylation. Activated PKR phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), which ultimately prevents delivery of methionyl-tRNA to the ribosome and blocks cap-dependent translation –6. In reMany poxviruses express two PKR antagonists, designated E3 and K3 in VACV. E3, the gene product of E3L, contains an N-terminal Z-nucleic acid binding domain and a C-terminal dsRNA binding domain. E3 inhibits PKR by binding to dsRNA and preventing PKR homodimerization ,12. K3, Our recent work has supported this hypothesis in the leporipoxvirus and capripoxvirus genera. We have demonstrated that capripoxvirus K3 orthologs inhibit PKR in a species-specific manner, strongly inhibiting sheep, goat, and human PKR, while only weakly inhibiting cow and mouse PKR . SimilarMost research on orthopoxviruses has focused on the prototypic poxvirus VACV as a model for poxvirus virology and host-virus interactions. However, very few studies have systematically investigated the activity of VACV orthologs in other orthopoxviruses. It is therefore unclear whether the activity of VACV proteins is representative of their orthologs in other orthopoxvirus. Orthopoxviruses such as CPXV and VARV represent substantial public health or bioterror threats, and human infections with orthopoxviruses are predicted to become more common as global immunity to poxviruses decreases in the population due to the cessation of the smallpox vaccination . TherefoIn this study, we compared inhibition of PKR from a panel of mammalian species by K3 orthologs from VACV, VARV, CMLV and CPXV-rat09 to determine whether the ability of VACV K3 to inhibit PKR from a given host was predictive of the ability for other orthopoxvirus K3 orthologs to inhibit this host antiviral protein. The results presented here show that the tested K3 orthologs exhibited distinct inhibition profiles and also that the phylogenetic relatedness of PKR was not a good predictor for how susceptible they were to inhibition by K3 orthologs. Mapping K3 residues that governed the species-specificity of these interactions revealed that multiple amino acid substitutions were necessary to convert a weak K3 ortholog into a better inhibitor of most tested PKRs. Furthermore, different amino acid combinations were needed to most effectively inhibit PKR from different species.Previous yeast-based and transfection assays indicated differential inhibition of PKR from several vertebrate species by VACV K3 ,9. In orkd cells with the K3L orthologs revealed comparable expression by Western blot  032, and CPXV 043 from the CPXV-rat09 strain, the latter of which is identical to multiple CPXV isolates from the D clade ,23, intoern blot . These tern blot . In addiern blot , we alsoern blot . Both asTo visualize whether the phylogenetic relatedness of a given PKR may be predictive of susceptibility to these K3 orthologs, we generated a phylogenetic tree using PKR sequences derived from 40 mammalian species. We included PKRs from several species that were not tested in the LBR assays to achieve better statistical support for the branches. We then mapped the relative sensitivities of the tested PKRs to each K3 ortholog obtained from multiple LBR assays using a susceptibility scale ranging from weakly sensitive to highly sensitive on a scale from 1 to 6, respectively . By thisko), all four viruses formed comparably sized plaques , is unknown [The strong activity of CMLV K3 against PKR from its natural host (camel) is in line with previous studies that showed that myxoma virus, sheeppox virus and goatpox virus inhibited PKR from their naturals hosts with high efficiency ,18. In c unknown .It is not clear why some poxvirus K3 orthologs are efficient inhibitors of their host’s PKRs, whereas others are not. One possible explanation is that different poxviruses may produce different amounts of dsRNA during infection. The formation of dsRNA is an important danger signal detected by multiple innate immune system sensors, and perhaps as a consequence, the amount of dsRNA produced during infection varies even between closely related orthopoxviruses. For example, vaccinia virus produces high amounts of dsRNA during infection, while MPXV and ectromelia virus (ECTV) produce lower amounts of dsRNA ,30. In aThe current knowledge about species-specific inhibition of PKR by K3 orthologs is summarized in Although VARV is a human-specific pathogen, this study and previous yeast-based assays demonstrated that the VARV K3L ortholog does not inhibit human PKR efficiently ,9. Howeva priori. Ultimately, it is likely that a combination of some or all of these interactions is necessary to permit a virus to replicate in a given host [In addition to the work on K3 orthologs, there is evidence that other poxvirus immune antagonists can have varying effects on the host. For example, while the E3 orthologs derived from swinepox virus and myxoma virus rescued E3L-deficient VACV in HeLa cells, the sheeppox virus E3 ortholog was not able to rescue this virus . Along tven host .Species-specific interactions of PKR with viral inhibitors have also been observed with cytomegalovirus (CMV) proteins. Human CMV TRS1 only inhibited human PKR but not PKR from old world monkeys, while the TRS1 ortholog from African green monkey (AGM) CMV inhibited AGM but not human PKR. Intriguingly, mutation of a single amino acid in human PKR (position 489), which determines susceptibility to human or AGM TRS1, is also involved in the weak sensitivity of human PKR to VACV K3 inhibition. Taken together, these data highlight that inhibitors from different pathogens can be affected by mutations in individual antiviral proteins .Large DNA viruses commit about half of their genes to counteract and redirect the host immune response. Because many viruses evolve with specific hosts, it can be assumed that a considerable portion of their encoded proteins will show some level of host-specificity, which ultimately contributes to their host range and virulence. Our study shows that orthologs of even closely related viruses can show dramatic differences in their interaction with the host innate immune system. Furthermore, the phylogenetic relatedness of PKR was not a good predictor for how well they were inhibited by K3 orthologs. These findings imply that research obtained with one specific virus and one specific host species cannot necessarily be generalized. Instead these interactions likely require experimental validation for other viruses and species.kd) (kindly provided by Dr. Charles Samuel [kd cells were maintained in medium containing 1 μg/ml puromycin (Sigma). RK13+E3L+K3L cell culture medium contained 500 μg/ml geneticin and 300 μg/ml zeocin (Life Technologies).HeLa , HeLa PKR-knockdown(s Samuel ), HeLa Ps Samuel ), RK13+Es Samuel , tert imSaII (forward primer) and NheI (reverse primer) sites, as described [All PKR and viral genes were cloned into the pSG5 expression vector (Stratagene) for transient transfection assay. Cloning of cow and sheep PKR, European rabbit PKR, mouse PKR, rat PKR, Syrian hamster PKR and guinea pig PKR was described previously . Orangutescribed . VARV C3escribed . All res4 Hela PKRkd cells per well were seeded in 24-well plates 16 hours prior to the experiment. Cells were transfected with 200 ng of the indicated PKR expression vector, the indicated amount of each K3L expression vector, and 50 ng of pGL3 firefly luciferase expression vector (Promega) using GenJet (Signagen) at a DNA to GenJet ratio of 1:2 following the manufacturer’s protocol. Empty pSG5 vector was used to maintain the DNA concentration at 200 or 100 ng where appropriate. 48 hours post-transfection cells were lysed with mammalian lysis buffer , then luciferin (Promega) was added following the manufacturer’s recommendations. Luciferase activity was measured using a GloMax luminometer (Promega). Experiments were conducted in triplicate in three independent experiments.Assays were performed as described  with theVACV Copenhagen strain VC2 and vP872  were kinPlaque assays were performed with confluent six-well plates of the indicated cell lines, which were infected with 30 plaque forming units (pfu) of each indicated virus, as determined on RK13+E3L+K3L cells. One hour post-infection, the medium was replaced with DMEM containing 1% carboxymethylcellulose (CMC). After 48 hours, cells were washed with PBS and stained with 0.1% crystal violet. The plates were imaged using an iBright Imaging System (Invitrogen).Multiple-cycle virus replication assays were carried out in confluent six-well plates of the indicated cells, which were infected with each indicated virus (MOI = 0.01). After 48 hours, cells and supernatants were collected and subjected to three rounds of freezing at -80°C and thawing at 37°C. Lysates were sonicated for 15s, 50% amplitude (Qsonica Q500). Viruses were titered by 10-fold serial dilutions on RK13+E3L+K3L cells. Infections and viral titer were performed in duplicate.5 Hela PKR-knock-down cells were seeded in six-well plates and transfected with 3 μg of the indicated K3-expressing plasmids following the GenJet protocol described above. After 48 h, cells were lysed with 1% sodium dodecyl sulfate (SDS) in PBS (VWR) and sonicated at 50% amplitude for 10 seconds twice. All protein lysates were separated on 12% SDS-polyacrylamide gels and transferred to polyvinyl difluoride  membranes. Membranes were blocked with 5% non-fat milk dissolved in TBST  for 1 hour. Blots were probed with a primary antibody (1:3000) directed against VACV K3 [To detect expression of K3 orthologs in transfected cells, 4 × 10 VACV K3 , or anti6 cells for 16 hours. Each cell line was then infected with the indicated virus (MOI = 5.0) for one hour. Inocula were then aspirated and replaced with fresh cell culture medium. After 6 hours, the cells and supernatants were collected and centrifuged at 800 RCF for 5 minutes. Cell pellets were lysed with 1% SDS in PBS (Gibco) and sonicated at 50% amplitude for 10 seconds twice. All protein lysates were separated on 10% SDS-polyacrylamide gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk powder in TBST for 1 hour. Membranes were probed with antibodies against total and phospho-eIF2α  at 1:3,000. Secondary antibodies were used as described above.Phosphorylated and total eIF2α was determined as follows: 6-well plates were seeded with 1 × 10The amino acid sequences of PKR from 40 different species  were ali


Isopropylbenzene (cumene) is commonly encountered in groundwater at petroleum release sites due to its natural occurrence in crude oil and historical use as a fuel additive. The cumene concentrations detected at these sites often exceed regulatory guidelines or standards for states with stringent groundwater regulations. Recent laboratory analytical data collected at historical petroleum underground storage tank (UST) release sites have revealed that cumene persists at concentrations exceeding the default cleanup criterion, while other common petroleum constituents are below detection limits or low enough to allow natural attenuation as a remediation strategy. This effectively makes cumene the driver for active remediation at some sites. An insignificant amount of research has been conducted for the in-situ remediation of cumene. Sulfate Enhanced Biodegradation (SEB) is evaluated in a field case study. The results from the field case study show an approximate 92% decrease in plume area following three rounds of SEB injections. An additional objective of this research was to determine the cumene concentration in fuels currently being used to determine future impacts. A review of safety data sheets from several fuel suppliers revealed that cumene concentrations in gasoline are reported typically as wide ranges due to the proprietary formulations. Several fuels from different suppliers were analyzed to determine a baseline of cumene concentration in modern fuels. The results of the analysis indicated that cumene accounts for approximately 0.01% (diesel) to 0.13% (premium gasoline) of the overall fuel composition. Cumene generally is considered to be of low human health toxicity, with the principal concern being eye, skin, and respiratory irritation following inhalation of vapors in an occupational setting, but it has been regulated in Florida at very low concentrations based on organoleptic considerations. Cumene (isopropylbenzene) is a volatile organic compound (VOC) consisting of an aromatic hydrocarbon with an aliphatic substitution . The comCumene is produced in high volumes in the United States with the majority of the chemical used as an intermediate in the synthesis of phenol and acetone . It is aCumene is commonly identified at petroleum-impacted sites when the released product type is unknown or is suspected to be used oil. In these situations, analysis according to Florida Administrative Code (FAC) Chapter 62-780, Table D is required. Table D includes analysis of priority pollutant volatile and extractable organics [Several petroleum release sites in Florida with documented cumene exceedances of state guidelines were identified through a search of the Florida Department of Environmental Regulation (FDEP) Oculus Database. Remediation data were available for only a few of the identified sites. Remedial techniques including In Situ Chemical Oxidation (ISCO), Sulfate Enhanced Biodegradation (SEB), Air Sparging/Soil Vapor Extraction (AS/SVE), groundwater extraction, or Monitored Natural Attenuation (MNA) were employed with varying degrees of success. Of the remedies reviewed, SEB was found to be among the most effective. This paper considers whether cumene can be remediated using sulfate-enhanced bioremediation and, in addition, assesses the foundation of the Florida criterion for remediation of cumene.High levels of cumene have been found in groundwater near industrial sites and in industrial effluents. Concentrations of 360  and 1581oc value of 3.45), chemical oxidation is not recommended for cumene treatment in groundwater [Pseudomonas fluorescens strain using cumene as its sole carbon source [Several techniques have been investigated for the removal and remediation of cumene. Due to its tendency to sorb to soils  is a proven remediation strategy for petroleum impacted groundwater. This remedial process works by applying an amendment of sulfate to an impacted medium that is currently using or can potentially use sulfate as a terminal electron acceptor (TEA). Native sulfate reducing bacteria (SRB) reduce the sulfate compound while simultaneously oxidizing the petroleum hydrocarbon. A general schematic showing the pathways of hydrocarbon degradation by bacteria under anaerobic conditions is presented in Site characterization is required to determine the identity and prevalence of the current electron acceptor. However, anaerobic bioremediation by sulfate reduction is the dominant terminal electron accepting process , and it Sulfate is highly water-soluble and does not readily sorb to soil particles, increasing the quantity of the TEA available to sulfate reducing bacteria . It is aGasoline and diesel samples were collected at gasoline service stations to establish a baseline of cumene concentrations in modern fuels. To maintain brand anonymity, the samples were labeled as one through five, with each number representing a different fuel supplier. A sample of regular (87 octane), mid-grade (89 octane), and premium (93 octane) gasoline was collected from each of the five fuel suppliers. In addition, a diesel sample was collected from Gas Stations #2, #4, and #5, and an ethanol-free sample was collected from Gas Station #3. The samples were placed into ANSI/ASTM/CARB-compliant and EPA-approved polyethylene gasoline canisters with sufficient volume  to minimize cross-contamination from other various fuel grades. Within 15 min of sample collection, a 2 mL aliquot from each gas can was transferred into a 9 mm clear glass autosampler vial using pipets. The sample vials were then immediately placed on ice and analyzed within 24 h of collection.The samples were analyzed utilizing a Shimadzu GCMS-QP2020 NX EI Gas Chromatograph Mass Spectrometer (GC-MS) equipped with a direct injection tower and a 30 m long by 0.25 mm inner diameter capillary column. The analysis was performed in general accordance with EPA Method 8260. It should be noted that a purge-and-trap autosampler and concentrator is typically used to analyze VOCs. However, EPA Method 8260 allows for the sample introduction to be performed by direct injection when volatile concentrations are in excess of 10,000 μg/L . This meSulfate Enhanced Bioremediation (SEB) was chosen as the remediation technology at a petroleum impacted site in Broward County, Florida (USA) after site characterization was performed. The site assessment and cleanup for the case study is state-funded under Florida’s Petroleum Restoration Program (PRP) and is being performed by an FDEP-contracted environmental consultant. In addition to the delineation of the petroleum contamination, site characterization also included analysis of various bioremediation and geochemical parameters such as common electron acceptors and metabolic byproducts in the plume and background monitoring wells. Specifically, the analysis included iron , manganese , alkalinity, biological oxygen demand (BOD), total organic carbon (TOC), nitrate, nitrite, total nitrogen, total Kjeldahl nitrogen (TKN), orthophosphorus, sulfate, sulfite, sulfide, heterotrophic plate count, hydrogen sulfide, carbon dioxide, and methane. Sulfite was analyzed in the field using a Hach SU-5 titration test kit. The remaining analysis was performed by a commercial laboratory using appropriate laboratory or EPA Methods. Sulfate was determined to be the active TEA based on the results. Following site characterization, the list of bioremediation parameters analyzed for subsequent remedial action monitoring events narrowed to include only alkalinity, sulfate, sulfite, and sulfide. VOCs, including benzene, toluene, ethylbenzene, xylenes, methyl tert-butyl ether (MTBE), and cumene were measured during each sampling event. The groundwater samples for VOC analysis were prepared using EPA Method 5030B and analyzed according to EPA Method 8260B. A commercial laboratory made the measurements on a GC-MS using a purge and trap unit before the instrument.2 with a vertical extent ranging from the top of the surficial aquifer at 1.5 m below land surface (bls), down to 10.7 m bls. Prior to the baseline analysis, cumene was the only contaminant identified at concentrations exceeding NADC standards. However, the baseline assessment also revealed benzene exceeding its respective NADC of 100 μg/L in one of the monitoring wells (170 μg/L). This field study was used as an example in Best Available Practice to remediate cumene contamination of groundwater.The baseline in-plume cumene concentrations ranged from 2.0 to 20 μg/L (average 9.74 μg/L) with NADC exceedances in four monitoring wells. The estimated plume area using baseline concentrations was 399.6 mSulfate was introduced into the impacted groundwater as an aqueous blend of magnesium sulfate  with water in an initial pilot study. The initial goal was to raise the in-plume sulfate concentrations from non-detect to approximately 200 mg/L. To accomplish this, approximately 568 L of an estimated 589 mg/L sulfate solution was injected at 12 separate points from 1.5 to 10.7 m bls . The solution was injected via a direct push drill rig using hollow steel rods and a pressure-activated injection probe. The injection was performed under low pressure  to ensure uniform distribution and minimal channeling. The depth to water and several geochemical parameters were monitored at various adjacent wells during the injection event to measure influence of the injection and any groundwater mounding effects.A total of three (3) injection events with sulfate were performed at the described remediation site. A site plan showing the current layout of the facility with monitoring well and injection point locations as well as other pertinent features is illustrated in An assessment of the health issues related to cumene exposure was conducted via a literature survey see . SpecifiThe cumene concentrations in fuels analyzed ranged from 125,802 (4-Diesel) to 1,323,250 μg/L (1-Premium). The margin of error is estimated to be +/− 3.5% with a confidence level of 95% based on the results of five separate runs on the same sample. Data from the GC/MS analysis of the fuels are shown in The results from the samples collected from Gas Station #1 show an increasing cumene concentration with increasing octane rating. For Gas Station #2, the cumene concentrations in the regular and midgrade are fairly similar, while the concentration in the premium fuel was significantly higher (approximately 41% higher). The diesel samples  exhibited the lowest concentrations of cumene. The chromatogram for the diesel samples  are uniq2). The first round of injections was conducted during the pilot test activities as described. The initial results showed a spike in contaminant concentrations, which is attributed to the mobilization of adsorbed contamination from the saturated soil source zone into the groundwater, followed by a steady decrease. Only minimal increases in sulfate concentrations in the plume were observed. The monitoring event performed after the 1st full-scale injection showed minimal to moderate increases in sulfate concentrations with slight cumene decreases. Major increases in in-plume sulfate concentrations (up to 370 mg/L) were observed after the 2nd full-scale injection. Cumene concentrations decreased in most monitoring wells immediately following the third injection with some wells showing slight increases. Large decreases in both sulfate and cumene concentrations were observed approximately 180 days following the third injection event. These concentrations reduced further in the subsequent monitoring event (sulfate concentrations at or below background concentrations). The regulatory GCTL criterion for cumene was then only exceeded in one monitoring well and the estimated plume size is approximately one-thirteenth of its original size . ConsisIn Florida, while there is no formal Drinking Water Standard for cumene, the assessment and management of cumene in groundwater are regulated under Chapter 62-780, Florida Administrative Code (F.A.C.) . USEPA a−1 mg/m3  is based on increased kidney weights in female rats and increased adrenal weights in male and female rats [Health-based groundwater screening levels such as those discussed here typically are based on toxicological guidance values—e.g., oral Reference Doses (RfDs) and inhalation Reference Concentrations (RfCs) that often are derived from animal studies. That is indeed the case for cumene. The oral RfD of 0.1 mg/kg·day is based on observed increased kidney weight in female rats, and the inhalation RfC of 4 × 10ale rats . ConservThe International Agency for Research on Cancer (IARC) classified cumene in Group 2B, possibly carcinogenic to humans , and theWith respect to potential off-gassing from groundwater to overlying soil and then into ambient air or into indoor air space of buildings over groundwater, according to USEPA’s Vapor Intrusion Screening Level (VISL) Calculator, cumene has a target groundwater concentration of 850 µg/L for protection against possible vapor intrusion exposure concentrations. That value is derived by back-calculating from safe indoor air screening levels established as protective health-based concentrations below which no further action or investigation is warranted .Analytical results from the samples collected showed cumene concentrations varying from 125,802 (4-Diesel) to 1,323,250 μg/L (1-Premium). The trend of increasing cumene concentration with increasing octane rating for Gas Station #1 fuels indicates that cumene is likely used as an octane boosting additive for the mid and premium grade gasoline blends at this station. The difference in concentrations between the mid-grade and premium gasoline for Gas Station #2 indicates that the compound is an octane booster for the premium gasoline only. The small variability of cumene concentrations in the fuels from Gas Station #3 suggests that the compound is either added to each grade at similar concentrations or it is naturally occurring in the source crude oil at those concentrations. The results from Gas Stations #4 and #5 also show very little variability in cumene concentrations between the different octane grades. However, the average cumene concentrations in Gas Stations #4 and #5  are much lower than that of Gas Station #3 . Additionally, cumene concentrations in 2–Regular and 2–Mid Grade are similar to cumene concentrations of all gasoline samples collected from Gas Stations #4 and #5. This may indicate that cumene concentrations in the range of 400,000–500,000 µg/L are present from natural sources. Concentrations reported in this study are considerably lower than values found in the literature (0.3% for gasoline and 0.86% for diesel). However, those values represent research conducted in the 1980s. Gasoline formulations change over time due to technological advancements and changing environmental regulations, which may explain the variability observed. Despite the lower concentrations in modern fuels, they are not insignificant given their persistence in the subsurface, coupled with restrictive regulatory criteria.The SEB successfully reduced cumene concentrations in this case study. The plume size was decreased by approximately 92%, and the site is on track to qualify for a clean closure (complete site rehabilitation). Clear increases in in-plume sulfate concentration were observed following the second and third injection events with the sulfate solution. In some cases, sulfate concentrations exceeded the sulfate secondary drinking water standard (250 mg/L). However, within two quarters following each injection event, sulfate concentrations had returned to background or undetectable quantities. This quick decline indicates that sulfate was likely the acting TEA. Based on the rate of TEA utilization, the time between injection events could have been decreased to approximately 6 months to condense the remediation time. All monitoring wells within the original plume extent tested below CTLs approximately 3.5 years after the pilot test injection. Currently, cumene is only detected in one shallow monitoring well, which is upgradient from the source area. That monitoring well was recently abandoned and replaced due to persistent observations of debris (roots) and intermittent turbidity issues noted during sampling. The replacement monitoring well has been sampled only once since installation, resulting in a slight cumene concentration. This level was likely caused by a disruption of the soils during the well installation activities. It is postulated that residual cumene mass adsorbed to saturated zone soils was temporarily released into the groundwater, similar to what was observed following the pilot test injection event. The estimated groundwater flow direction and a comparison of current and former plume extents (based on the GCTL for cumene) is illustrated on Due to its success at this site, pilot-scale SEB is being implemented at a few other sites in South Florida to evaluate its effectiveness for remediation of other VOCs and Polycyclic Aromatic Hydrocarbons (PAHs). Initial results indicate that SEB is successfully remediating other VOCs such as benzene, toluene, ethylbenzene, xylenes, and some cumene isomers, but it may not be appropriate for general PAH-driven remedial activities.It is likely that the added sulfate stimulates the growth of sulfate-reducing bacteria, which are the primary cause of the cumene breakdown. More research needs to be conducted on the bacterial genomics of specific groundwater species that cause the cumene breakdown.Because of the limited human health concern regarding cumene, and because the default groundwater cleanup guideline is not a health-based value, potentially responsible parties and FDEP remedial project managers should carefully evaluate risk-based options when cumene in groundwater is the driving substance of a remediation project. Perhaps the Florida Department of Environmental Protection should re-evaluate the very low concentration used to trigger remedial activities, especially when the concentrations of other contaminates do not exceed remediation guidance. Since cumene is degraded by naturally occurring, anaerobic bacteria, remediation on sites with low concentration, even above the regulatory limit, should be evaluated using risk-based criteria. At locations where human exposure is unlikely, natural attenuation should be given consideration as a remedial strategy.Cumene (Isopropylbenzene) is a naturally occurring substance in unrefined petroleum and is used as an additive in medium and high-grade gasoline to enhance octane. It is regulated in water in 14 of 50 states with action standards ranging from 5 to 840 µg/L. Because of the paucity of data with regard to impacts on human health, the clean-up guidelines used by the states that have them are not based on public health considerations. Cumene is considered to have low toxicity to humans but is regulated at very low concentrations in Florida (8 µg/L).An effective means of remediating cumene in groundwater is Sulfate Enhanced Biodegradation (SEB). Addition of sulfate to groundwater promotes growth of various anaerobic bacteria that cause breakdown of cumene into harmless constituents. Typical remediation methods for other gasoline components are not effective for use on cumene.Based on a generally low potential impact to human health, a different, risk-based approach to mandating remediation should be considered, especially at sites where potential contact with humans is minimal. Most groundwater in Florida is anoxic and contains the specific types of anaerobic bacteria used in the SEB remediation strategy. Therefore, natural attenuation is a remediation strategy that should be considered in low risk sites in lieu of performing active remediation.


Cryptococcus gattii (C. gattii) has been reported in Canada and the United States, but there were sparsely-reported cases of C. gattii in China. Our interest in occurrence, clinical manifestation, laboratory identification and molecular characterization of Chinese C. gattii strains leads us to this research.Infection, even outbreak, caused by Cryptococcus neoformans (C. neoformans), eight strains were re-identified as C. gattii. Multi-locus sequence typing (MLST) showed genotype VGI accounted for the most (6 / 8), the other two strains were genotype VGII (VGIIa and VGIIb respectively) with 3 specific spectra of molecular weight about 4342, 8686, 9611 Da by MALDI-TOF MS. The minimal inhibitory concentrations (MICs) of Fluconazole with Yeast one was 2~4 times higher than that with ATB fungus 3 and MICs of antifungal agents against VGII strains were higher than against VGI strains. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins was directed to Golgi complex.Out of 254 clinical isolates, initially identified as C. gattii in China occurred sparsely. Genotype VGI was predominant but VGII was more resistant to antifungal agents. There was significant difference in protein expression profile between isolates of VGI and VGII C. gattii.Infection by  Cryptococcus gattii (C. gattii), one of the most common pathogenic cryptococcal species, primarily infects immunocompetent hosts, in contrast to Cryptococcus neoformans (C. neoformans), which mainly infects immunocompromised individuals [C. gattii infection was restricted geographically to tropical and subtropical regions [C. gattii infection, to investigate clinical and microbiological characterization, and to analyze molecular features of the Chinese isolates.ividuals , 2. Prev regions –3. Howev regions , 5. The  regions . The objC.gattii by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with scores from 2.043 to 2.275, otherwise they were all identified as C.neoformans by VITEK 2 compact as seen in Table C. gattii isolates were characterized by diverse spectra in the range between 2000 and 10,000 Da. Three specific spectra with molecular weight of about 4342, 8686, 9611 Da could be seen in two isolates of C. gattii VGII, they were absent in six isolates of C. gattii VGI, as shown in Fig.C. gattii VGI and VGII as shown in Fig.C. gattii were listed in Table Among 254 collected Cryptococcal strains, eight showed blue pigmentation on canavanine glycine bromothymol blue (CGB) agar and were characterized as C. gattii VGI and two strains of C. gattii VGII as indicated in supplementary Table C.gattii genotype VGII could be subtyped into VGIIa and VGIIb respectively by further comparison with Vancouver Island reference stains of R265 and R272, as in supplementary Table MLST confirmed six strains of C. gattii were isolated from eight patients. Two patients with isolate numbers of 2 and 6 were from Hainan and Yunnan Province, which were recognized as tropical areas. Three patients with isolate number 3 (from Chongqing city), 4 and 7 (from Fujian Province) were recognized as being from subtropical area. The other three patients with isolate numbers of 1, 5 and 8 were from Shandong Province, Inner Mongolia Autonomous Region and Henan Province, which were recognized as temperate regions. Information of patient infected with isolate number 3 was unavailable, the other 7 patients were immunocompetent young male adults, with age range of 21~60 years old. Their clinical and laboratory information were summarized in Table As shown in Table C. gattii were created. A total of 3628 proteins were identified, of which, 2436 proteins were found in both VGII and VGI C. gattii, however, C. gattii genotype VGI and VGII possessed 774 and 418 specific proteins respectively. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed in C. gattii VGI and VGII as shown in Fig.In this study, the proteome expression profiles of two strains of VGII and four strains of VGI The information of GO term and KEGG metabolic pathway analysis indicated that the enrichment of differential expressed proteins between VGI and VGII was mainly directed to Golgi apparatus, Golgi membrane and Golgi vesicle as shown in Table C. neoformans and C. gattii are two important fungal pathogens for humans and animals. Both of them are round with capsule and positive urease test. These routine laboratory identification methods are unable to distinguish the two species [C. gattii from China [C. gattii infections may have been underestimated because most laboratories utilize ink staining and biochemical methods, like VITEK 2 compact, for routine identificatio. CGB agar was recommended to identify C. gattii from C. neoformans due to its convenience and low cost [C. gattii were not only correctly identified but also grouped by MALDI-TOF MS with Bruker software 3.0. Of course, eight strains was not enough and more strains should be studied to obtain the spectrum difference between groups of C. gattii. species . Althougom China –10, the low cost , but it C.gattii isolates were susceptible to 5-Flucytosine, Amphotericin B, Itraconazole, Voriconazole and Posaconazole, only one strain was resistant to Fluconazole with MIC of 128 by Yeast one. However, interpretation of MICs of antifungal agents including Fluconazole for category of “susceptibility” or “resistance” was hampered by the lack of clinical breakpoints for C. gattii. Some previous studies have shown that C. gattii may be less susceptible to antifungal agents compared with C. neoformans. In a study from Taiwan, C. gattii was less susceptible to 5-Flucytosine and amphotericin B, and in Spain, MICs of Fluconazole, Voriconazole and Posaconazole to C. gattii were significantly higher [C. gattii has rarely been studied [C. neoformans and C. gattii isolates from Europe, USA, Australia, Brazil, Canada, India and South Africa. ECVs of Amphotericin B (0.5~1 μg/ml), 5-Flucytosine (4~16 μg/ml), Fluconazole (8~32 μg/ml) and other azoles varied similarly by molecular type for both C. neoformans and C. gattii [The results of antifungal susceptibility test by two kits were in good agreement, except that MICs of Fluconazole were 2~4 times higher by Yeast one than by ATB fungus 3. All the y higher –13. The  studied , our dat studied –17. Howe. gattii , 16.C. gattii have been detected, they were VGI, VGII, VGIII and VGIV. Lockhart and colleagues had reported that VGII and VGIII were the most-frequently identified isolates in America, VGIV was almost exclusively in Africa, and VGI predominated in Europe, Australia and Asia [C. gattii, as an important pathogen, caused outbreak in British Columbia, Canada and the Pacific Northwest, the United States. But 8 strains in our study were pathogens causing sporadic infections according to the strain origination and their isolation time.Up to now, four genotypes of and Asia . C. gattC. gattii infection occurred regionally and domestically. They were immunocompetent young male adults with age range of 21~60 years old, which was considered as the reason for increased exposure to environmental sources and increased chance for infection [.. Most patients had headache and fever, three patients showed neck stiffness, positive Kernig’s sign and meningeal lesions, meningeal enhancement or lacunar infarction by brain MRI. Most of them also demonstrated irregular nodule, consolidation and mass by lung CT, which was consistent with the research made by Ngamskulrungroj who indicated that C. gattii could cause fatal lung infection [All the patients in our study had no recent travel history, the nfection .. Most pnfection .C. gattii VGII strains were more virulent than VGI strains and VGIIa were even more virulent than VGIIb independent of their clinical or environmental origin [C. gattii VGI more than C. gattii VGII on the one hand, and on the other hand, two of them were infected with C. gattii VGII which showed sequence mutations in the gene location of RAS1 and FTR1 compared with reference strains of R265 and R272 respectively. Whether the mutations were relevant to virulence decrease, further work needs to be done.It was reported that host response varied based on the genotype of the organism and concomitant illnesses . There wl origin . In our C. gattii VGI and VG II was mainly located on the organelles associated with the Golgi body, which meant energy metabolism of C. gattii might be involved in the difference of pathogenesis mechanism for different genotype of C. gattii. Study on the secretory protein and the secretory vesicles of the two genotypes of C. gattii would be beneficial to the understanding of the pathogenesis of C. gattii.Our study showed significant difference in protein expression spectra between VGI and VGII. GO is a very important tool of biological information. By establishing a set of controlled words with dynamic form, the attributes of genes and gene products in organisms can be described comprehensively. KEGG is the main public database with which the most important biochemical metabolic pathways and signal transduction pathways can be determined. Our results suggested that the differential protein of C. gattii genotype VGI was predominant in China and C. gattii genotype VGII was more resistant to antifungal agents. C. gattii VGII isolates possessed obvious protein peaks with molecular weight of approximate 4342, 8686, 9611 Da. The full protein spectrum data of C.gattii were provided for the first time, a total of 3628 proteins were identified, 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins between VGI and VGII was mainly directed to Golgi complex. It will lay a foundation for better understanding and further research on the pathogenic mechanism for different genotype of C. gattii.In spite that only 8 strains were available for analysis, our results demonstrated that C. neoformans by biochemical methods were collected from seven hospitals in China, they were all stored at − 80 °C. All the isolates were sub-cultured onto Sabouraud dextrose agar medium at 25 °C for 48~72 h and identified by VITEK 2 Compact . Each isolate was also inoculated on canavanine-glycine-bromthymol blue (CGB) agar [A total of 254 clinical isolates initially identified as GB) agar  at 25 °CC.neoformans were included as outgroup control.Isolates were re-identified and analyzed by MALDI-TOF MS . Briefly, one single colony was smeared directly on the MALDI-TOF MS analysis plate and formic acid was added. After 3~5 min, matrix was added and the plate was put into the MALDI-TOF MS instrument with MALDI Biotyper software 3.0  for species identification and dendrogram analysis. Two strains of C. gattii was then extracted using the TianGen® TIANamp Yeast DNA Kit  complying with the manufacturer’s instruction, seven genes of unlinked loci were amplified, including six housekeeping genes  and one non-coding region gene (IGS1) [C.neoformans/C.gattii species complex (http://mlst.mycologylab.org) one by one. A sequence type (ST) number and seven allele type (AT) numbers were given to each isolate. New AT and ST number will be assigned for new sequence. To differentiate VGII subtypes, twelve unlinked genes  were also amplified and sequenced according to the method of Fraser et al. [Genomic DNA of each isolate identified as e (IGS1) , bi-direr et al. , they weCandida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 were used for quality control.Antifungal susceptibility test was carried out by both ATB fungus 3  and Yeast one  following their instructions. C. gattii was reviewed retrospectively, including age, sex, underlying diseases, symptoms, imaging findings, laboratory examinations and antifungal therapy.Under authorization of Medical Record Department, Clinical information of patients infected by C. gattii were selected for proteome analysis. All the isolates were sub-cultured onto Sabouraud dextrose agar medium, incubated at 25 °C for 48~72 h , the proteins were extracted from the strains, The protein concentration was 0.5 μg/μl, after being separated by SDS-PAGE electrophoresis , the proteins were hydrolyzed and analyzed. Original mass spectrum files of 6 strains were imported into Maxquatt software (version 1.3.0.5) for analysis. The database source was for C.gattii. in Uniprot (http://www.geneontology.org/). Plus or minus 15 ppm of polypeptide molecular weight, or missing 2 cutting sites were set as Maxquatt parameters, false discovery rate (FDR) for polypeptide identification was set as 0.01. A standard of p < 0.05 and 2-fold expression were considered as different proteins. The enrichment and pathway analysis was carried out by using the GO plot package of R language and KEGG online tool . The bubble diagram was drawn according to the R language ggplot2 package.According to the regional distribution and the integrity of clinical data of the strains, six strains (4 of VGI and 2 of VGII) of Additional file 1: Supplementary Table 1: MLST profiles and genotype of 8 C. gattii strains. Supplementary Table 2: MLST profiles of 16 gene loci for 2 C. gattii strains with genotype VGII. Notes: R265 and R272 were Vancouver Island reference strains for C. gattii genotype VGIIa and VGIIb respectively; / represented that data was not available; GenBank accession numbers for multilocus sequence typing alleles were: CAP59–1, DQ096432; CAP59–2, DQ096433; GPD1–1, DQ096377; GPD1–6, DQ096382; IGS-4, DQ096314; IGS-10, DQ096319; PLB1–1, DQ096343; PLB1–2, DQ096344; SXI1α-18, DQ096308; SXI1α-19, AY973646; TEF1–7, DQ096364; TEF1–5, DQ096362; FTR1–1, DQ096448; FTR1–2, DQ096449; CBP1–1, DQ096435; CBP1–2, DQ096436; ICL1–1, DQ096458; ICL1–2, DQ096459; HOG1–1, DQ096456; TOR1–1, DQ096470; STE7–1, DQ096467; STE7–2, DQ096468;TRR1–1, DQ096472; TRR1–2, DQ096473; RAS1–1, DQ096464; RAS1–2, DQ096465; PAK1–1, DQ096461; PAK1–2, DQ096462.


To estimate the direct and indirect costs of diabetes mellitus care at five public health facilities in Kenya.We conducted a cross‐sectional study in two counties where diabetes patients aged 18 years and above were interviewed. Data on care‐seeking costs were obtained from 163 patients seeking diabetes care at five public facilities using the cost‐of‐illness approach. Medicines and user charges were classified as direct health care costs while expenses on transport, food, and accommodation were classified as direct non–health care costs. Productivity losses due to diabetes were classified as indirect costs. We computed annual direct and indirect costs borne by these patients.More than half (57.7%) of sampled patients had hypertension comorbidity. Overall, the mean annual direct patient cost was KES 53 907  . Medicines accounted for 52.4%, transport 22.6%, user charges 17.5%, and food 7.5% of total direct costs. Overall mean annual indirect cost was KES 23 174  . Patients reporting hypertension comorbidity incurred higher costs compared with diabetes‐only patients. The incidence of catastrophic costs was 63.1%  and increased to 75.4%  when transport costs were included.There are substantial direct and indirect costs borne by diabetic patients in seeking care from public facilities in Kenya. High incidence of catastrophic costs suggests diabetes services are unaffordable to majority of diabetic patients and illustrate the urgent need to improve financial risk protection to ensure access to care. Kilifi is located on the coast of Kenya, and a high burden of stroke and heart failure has been described in this area.Six public health care facilities were purposively selected in consultation with county health officials in respective counties to generate a sample of facilities with different workloads, plus the location of the clinics relative to the communities served. However, due to the 150‐day nation‐wide nurses' strike at the time of data collection,2.2Zα/2 is the critical value of the normal distribution at α/2 , Zβ is the critical value of the normal distribution at β , P is the expected true proportion of DM in the population in Kenya of 10% (0.10), and e is the desired standard size of standard error around the estimated proportion of 5% (±0.05).The target enrolment was 282 patients for a sample size sufficient to obtain an estimate of DM patient costs based on the formulae by KirkwoodEvery DM patient receiving treatment and available at participating facilities during data collection was approached to participate in the study. Patients were eligible if they self‐reported DM diagnosis, had received treatment for a minimum of 6 months after diagnosis and were more than 18 years of age. Consenting patients were selected based on meeting the eligibility criteria and the order of arrival at the clinic. Respondents were asked to report on their health service use, associated costs, income, and coping mechanisms if they undertook any of the following to meet DM care costs: borrowing (having taken a loan), selling household items or assets , and use of savings.2.3The cost‐of‐illness approach was used to document patient costs.To annuitize sick visit costs, we summed up costs incurred during current care visit and any reported outpatient visit costs that occurred due to DM in the last 4 weeks prior to the study then multiplied by 13 (assuming there are 52 weeks in a year). On the other hand, to annuitize costs in other care‐seeking episodes described in Table For each of the care‐seeking episode, two broad cost categories were estimated: direct OOP costs and indirect (productivity losses) costs for both patients and their caregivers. Direct health care costs included any charges levied for medicines and user fees, i.e., registration, consultation, and laboratory services. Direct non–health care costs included transport costs to and from a health provider and any costs associated with food or accommodation while seeking care. For this analysis, OOP costs were defined as the sum of direct health care and direct non–health care costs. The analysis was restricted to patients who reported any OOP costs for each care‐seeking episode.Indirect costs were estimated based on the total hours lost while seeking care as well as the cost of illness‐related to lost productivity for both patients and their caregivers, assuming that these hours would have been used for productive activity in the absence of DM.Income was estimated by asking detailed questions about income categories, including patient income, income for household members, welfare payments, and government assistance. As a measure of financial risk protection, we compared total direct costs incurred in the overall sample and by socio‐economic status, against annual household income and total direct costs excluding transportation costs and defined costs as catastrophic if they exceeded 10% of annual household income.2.4Data were double entered to enhance data quality. The two data sets were compared to eliminate data entry errors. Consistency and range checks were used to ensure the completeness of data. Data were analysed using STATA 14.0 . Frequency counts and percentages were used to describe patient demographic and socio‐economic variables. Due to skewed nature of costs data, mean and median values were reported for all cost estimates as a measure of central tendency and 95% confidence intervals (CI) and interquartile range (IQR) were reported. Cost results are presented in KES and US Dollars (US$). Cost in KES was converted to US$ using the following exchange rate: US$ 1 = KES 102 .2.5Ethical approval for the study was obtained from the Scientific Ethics Review Unit (SERU) of KEMRI (Ref: KEMRI/SERU/CGMR‐C/041/3270) and from the County Department of Health in both study sites. Written consent was obtained from each participant. Facility managers were informed about the study and granted permission to access clinics and patients.33.1Overall, 163 patients were interviewed: 92 (56.4%) from Bungoma and 71 (43.6%) from Kilifi. The mean age in the sample was 58.9 years, and 58.9% of the respondents were female. More than half of the patients reported hypertension comorbidity (57.7%), were not enrolled to any health insurance scheme (68.1%), and slightly under half were diagnosed with either DM or hypertension (HTN) more than 5 years ago (47.9%)  of patients reported a sick visit, i.e., sought care out of their scheduled clinic appointments a month before the survey. Similarly, 29% (n = 20) of diabetes‐only patients and 41% (n = 39) of comorbid patients reported a sick visit. Mean annual costs for medicines attracted the highest direct cost in the overall sample—KES 15 340.8   mainly driven by the contribution of medicine costs for comorbid patients—KES 17 880  . However, among diabetes‐only patients, user charges attracted the highest mean annual cost—KES 12 733.5   (Table 9 of pati3.2.2Twenty‐two percent (n = 37) of the patients in the study reported an inpatient admission in the past year with each admission lasting a mean of 1.6 days . User charges constituted the highest direct costs attracting an estimated KES 16 718.8  , KES 18 568.6  , and KES 15 273.9   mean annual costs in the overall sample, diabetes‐only patients, and comorbid patients, respectively. Additionally, direct costs were higher than indirect costs in all the three groups (Tables 3.2.3The median number of routine medicines prescribed to diabetes‐only patients was 2 IQR 1‐2) and 3 IQR, 3‐4) for comorbid patients. The most expensive antidiabetic regimen was metformin and insulin   while metformin, glibenclamide, and enalapril combinations were the most expensive medicines prescribed to comorbid patients—mean annual cost KES 18 635.4   (Table ‐2 and 3  for como3.2.4The main routine diagnostic tests reported by diabetes‐only patients were fasting blood sugar (FBS) and weight—mean annual cost KES 1499.4   while comorbid patients reported FBS and blood pressure, mean annual cost KES 4518.6  , and echocardiogram and kidney function tests, mean annual cost KES 3335.4  . The mean annual direct costs for diagnostic/laboratory tests were generally higher for diabetes‐only patients  attended scheduled clinic visits monthly. Medicines attracted the highest cost in the overall sample, mean annual cost KES 10 159.2  , and in the comorbid patients, mean annual cost KES 12 839.2  . However, user charges attracted the highest cost, mean annual cost KES 6362.2   in the diabetes‐only group during scheduled clinic appointments. In addition, transport costs accounted for 23.2%, 26.5%, and 21.3% of total direct OOP costs in the overall sample, diabetes‐only, and comorbid patients, respectively  —was higher than the average indirect annual costs—KES 23 174.4  —in the overall sample. Of note, medicine costs in the overall sample, in the diabetes‐only patients, and in the comorbid patients attracted the highest costs. This was closely followed by transport costs in the overall sample and in the comorbid patients while user charges were the second highest cost category after medicines in the diabetes‐only group (Tables 3.3Costs for DM care services was catastrophic to three quarters n = 123), 75.5%  of patients at the 10% annual household income threshold. Moreover, comorbid patients (n = 94) realized higher catastrophic costs, 79.8% , compared with diabetes‐only patients (n = 69), 69.6% . Alternatively, when transport costs were excluded, (n = 103) 63.2%  of patients incurred catastrophic costs. Among patients experiencing catastrophe, patients in the lowest wealth quintile incurred higher direct costs with few resources to meet the health care costs  working days. Forty‐five percent of the respondents reported disrupted social life due to DM, affecting sexual life (n = 11), job loss (n = 48), divorce (n = 4), and separation from spouse (n = 6).4This study has estimated patient costs for adults with DM that sought care at five public health care facilities in two counties in Kenya. The study's main finding is that patient cost for DM are driven by medicine expenses. Similar findings have been reported by recent studies in Kenya and South Africa.The incidence of catastrophic costs documented in this study is arguably high and suggests that DM care in the sampled health facilities is unaffordable to majority of patients, especially those in the lowest wealth quintile whose capacity to pay is limited compared with those in higher socio‐economic group. This is a concern given the high poverty rates in Kenya (36.1%) and that only 19% of Kenya's population have a form of health insurance.Our results indicate that transport cost offers an access barrier to DM patients given that it takes a significant proportion of total direct costs in all care‐seeking episodes. In part, the high transport costs reported in this study can be attributed to poor quality of care in public health care facilities. For instance, 48.9% of the patients reported lack of medicines and diagnostic facilities as a reason for not visiting nearest facilities. This phenomenon has been observed from studies in Uganda and Zambia.The indirect costs due DM care in this study are noteworthy. For instance, of the 163 patients enrolled in the study, 48 (29.5%) had to stop working because of DM and 54 (33%) reported a median loss of 21 working days over the past 90‐day period, which is equal to 1 month's wage in the informal sector in Kenya. Similarly, the overall mean indirect costs in all care‐seeking episodes was KES 23 174 (US$ 227.2) and were primarily contributed by long waiting times at health facilities. It has been shown that long‐waiting times while receiving care reduces demand for chronic care services.5Our study had some limitations. Due to the recall periods, results may be subject to recall bias. Our study only focused on those who utilized health services, thus excluding those with undiagnosed diabetes who may still incur costs due to symptoms associated with diabetes. Also, costs reported in our study could be potentially overestimated since patients were selected purposefully. Consequently, our findings are not nationally representative. Also, our study relied on the diagnosis reported by patients, hence could not distinguish between type 1 and type 2 diabetes. Use of an official minimum wage to estimate productivity losses for all patients could have potentially overestimated indirect costs among patients who were unemployed prior to their illness or underestimated indirect costs among those who were employed.Edwine Barasa, Anthony Etyang, Kenneth Munge, Jane Mbui, Zipporah Bukania, Fredrick Kirui, and Andrew Obala conceived the study. Robinson Oyando, Martin Njoroge, Antipa Sigilai, Kenneth Munge, Peter Nguhiu, and Edwine Barasa contributed to the development of data collection tools. Robinson Oyando collected the data. Robinson Oyando and Edwine Barasa analysed the data. Robinson Oyando developed the first draft of the manuscript. All authors contributed to writing subsequent versions of the manuscript.The authors declare that they have no competing interests.This work is funded by a KEMRI internal research grant (IRG‐04) awarded to Anthony Etyang. Additional funds from a Wellcome Trust research training fellowship (#107527) awarded to Edwine Barasa, a Wellcome Trust International Master's Fellowship (#214622) awarded to Robinson Oyando, and a Wellcome Trust core grant (#092654) awarded to the KEMRI‐Wellcome Trust research program supported this work.


The COVID-19 pandemic has generated many urgent questions on the origin, trajectory, and host preference of its causative betacoronavirus SARS-CoV-2, as well as renewed focus on other potentially zoonotic viruses. Several species of birds and wild bats can serve as reservoirs and/or mechanical vectors for many infectious viruses including influenza-A, SARS-CoV, MERS, and Ebola. Although substantial progress has been made, there are still major gaps in understanding the emergence, transmission, and adaptation of zoonotic avian- and bat-borne viruses. A major challenge is the dearth of suitable infection and immunity models. Extrapolating data from infection studies in human cell lines or rodents is limiting, as evolutionarily optimized immune factors function differently in non-hosts.Campbell and Magor). In chickens, key innate immune genes, such as IRF3 and RIG-I, appear to have been lost  and NF-κB, respectively. IFN induces production of hundreds of “interferon-stimulated genes” (ISGs), limiting virus replication until the adaptive immune response can “clean-up.” Rapid initiation of innate immune responses depends on viral recognition by pattern recognition receptors (PRRs) which are expressed in tissues where the virus replicates. Despite the high degree of evolutionary conservation, and assumed similarity in function, there are significant differences between the immune gene repertoires of birds and mammals and bats and other mammals. Ducks utilize many of the mammalian PRRs  and their downstream adaptors to detect and inhibit the replication of influenza viruses . The full-length PA-X and PB1-F2 proteins in a recombinant 2009 pH1N1 virus have been reported to enhance viral replication in vitro, enhancing viral virulence in vivo mainly through simultaneously mediated host innate immune response .Differences in IFN-inducing capacity between virus strains is an important parameter contributing to the host response to viral infection and virulence. Influenza-A virus circulates in multiple hosts causing economic burden in the poultry industry, human epidemics, and occasionally pandemics. Influenza-A virus contains eight negative-strand RNA segments, which are thought to encode 10 viral proteins as well as seven newly identified proteins such as PB1-F2, PA-X whose role is not entirely clear  is another economically important (but not zoonotic) virus for the poultry industry. Very virulent strains of IBDV (vvIBDV) emerged in the 1980s, causing up to 60% mortality in some commercial flocks for reasons that are poorly understood . DulwichReddy et al.).The successful control of virus infection requires the coordination of both the innate and adaptive immune systems. During chronic viral infections the increased level or duration of stimulation of virus-specific CD8 T-cells leads to non-functional state called T-cell exhaustion . Recent Pteropus alecto) (Clayton and Munir). Sequencing of bat genomes suggests that evolution of “metabolically costly” flight imposed adaptations on their innate immune-, DNA damage- and inflammatory-response systems . Studies have inferred a pattern of constitutively expressed IFN-α in unstimulated Pteropus alecto cells, which may provide a “switched-on” defense mechanism that blunts virus replication and pathogenesis (Giotis).Current knowledge on bat IFN responses is rudimentary, with most of this obtained in studies of the Australian flying fox . Furthermore, the aftermath of SARS-CoV-2 internalisation is governed by a complex and largely unknown network of host-pathogen interactions. Transcriptomic analysis of human alveolar and bronchial epithelial cells confirmed that the CSF1 gene, a known target of the microRNA miR-1207-5p, is over-expressed following SARS-CoV-2 infection . CSF1 enhances macrophage recruitment and activation and its overexpression may contribute to the acute inflammatory response observed in severe COVID-19.Full-length genome sequences obtained from COVID-19 patients demonstrated that SARS-CoV-2 shares 79.5% sequence identity to SARS-CoV BJ01 strain and a 96% sequence identity to the bat coronavirus RaTG13, suggestive that bats are the original source of the virus . The recIn conclusion, recent virus outbreaks confirmed the inextricable nature of human and animal health and disease. Interdisciplinary approaches are urgently needed to assess and prevent the spillover of avian- and bat-borne viruses using an organismal biology approach and focusing on viral ecology, diversity, and interactions with the host.EG wrote the first draft and JS and DM edited and commented on the draft. All authors contributed to the article and approved the submitted version.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.


Besnoitia besnoiti, is a chronic and debilitating cattle disease that notably impairs fertility. Acutely infected bulls may develop respiratory signs and orchitis, and sterility has been reported in chronic infections. However, the pathogenesis of acute disease and its impact on reproductive function remain unknown.Bovine besnoitiosis, caused by the apicomplexan parasite B. besnoiti infection. Acute infection was confirmed by serological techniques (IgM seropositive results and IgG seronegative results) and subsequent parasite detection by PCR and histological techniques.Herein, we studied the microscopic lesions as well as parasite presence and load in the testis  of seven bulls with an acute The most parasitized tissue was the scrotal skin. Moreover, the presence of tachyzoites, as shown by immunohistochemistry, was associated with vasculitis, and three bulls had already developed juvenile tissue cysts. In all animals, severe endothelial injury was evidenced by marked congestion, thrombosis, necrotizing vasculitis and angiogenesis, among others, in the pampiniform plexus, testicular parenchyma and scrotal skin. Vascular lesions coexisted with lesions characteristic of a chronic infection in the majority of bulls: hyperkeratosis, acanthosis and a marked diffuse fibroplasia in the dermis of the scrotum. An intense inflammatory infiltrate was also observed in the testicular parenchyma accompanied by different degrees of germline atrophy in the seminiferous tubules with the disappearance of various strata of germ cells in four bulls.This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanent, which is supported by the severe lesions observed. Consequently, we hypothesized that testicular degeneration might be a consequence of (i) thermoregulation failure induced by vascular lesions in pampiniform plexus and scrotal skin lesions; (ii) severe vascular wall injury induced by the inflammatory response in the testis; and (iii) blood-testis barrier damage and alteration of spermatogenesis by immunoresponse. Besnoitia besnoiti [Bovine besnoitiosis is a cattle parasitic disease caused by the cyst-forming apicomplexan protozoan besnoiti . The disbesnoiti  alerted besnoiti . Besnoitbesnoiti , 5.Several authors have reported that beef cattle are more susceptible to the infection than dairy cattle that could be attributed to risk factors associated with the management husbandry system. In fact, in beef cattle herds males appeared to show acute clinical signs more frequently and higher mortality rates as they might be more exposed than females due to natural mating , 7. ReceClinical besnoitiosis outcome occurs in two sequential phases. First, an acute febrile phase appears due to the fast-replicating tachyzoite stage in endothelial and mononuclear cells. This phase is characterized by generalized oedema and ocular and nasal discharge . Bulls mIn the affected herds, most animals remain subclinically infected and only a small number develop noticeable clinical signs compatible with either acute or chronic besnoitiosis that represent the tip of the iceberg , 4. ChroB. besnoiti infection. In addition, the pathogenic mechanisms that govern acute disease were also discussedThe objective of this work was to determine the microscopic lesions along with their impact on reproduction function and their association with parasite stage and load in the testicles of bulls with an acute n = 4), Limousin (n = 1) and breed unknown (n = 2)  diluted 1:10,000 in phosphate-buffered saline containing 0.05% Tween 20 (PBST) was included. Control sera used in the ELISA came from an experimental infection carried out in calves. The positive control serum was composed of three sera from calves that were intravenously inoculated with 108 tachyzoites of Bb-Spain 3 isolate and collected at 4 days post-infection (pi). The negative control serum comprised a pool of three sera from the negative control group [An IgM BbSALUVET ELISA 2.0 was employed following a previously described procedure . This ter et al.  to detecol group . The optSera were analysed by BbSALUVET ELISA 2.0  to discrTo discriminate between acute and chronic infection, an avidity ELISA was performed when specific IgGs were detected by BbSALUVET ELISA 2.0. The test was carried out as previously described by Diezma-Díaz et al. . Sera weSALUVET tachyzoite-based western blot (WB) was performed under non-reducing conditions in 12.5% polyacrylamide gels  to confiDNA extraction of the different tissues collected  was carried out using the Maxwell® 16 Instrument  with the Maxwell® 16 Tissue DNA Purification Kit (Promega) . The DNABesnoitia spp. DNA was detected by ITS1 rDNA PCR [rDNA PCR . The cycrDNA PCR .in vitro cultured B. besnoiti Bb-Spain 1 tachyzoites and PCR grade water were used as the positive and negative controls, respectively.The amplified products were visualized after electrophoresis on a 1.5% agarose gel containing 0.1 μl/ml GelRed™ Nucleic Acid Gel Stain . DNA extraction and PCR were performed in separate laboratories under biosafety level II conditions  to avoid cross contamination. DNA extracted from Besnoitia spp. DNA was performed according to Cortes et al. [Besnoitia PCR-positive samples were further analysed by qPCR.The qPCR assay for the quantification of s et al.  and Freys et al. . The fors et al. . In eachs et al. . Only BeRoutine paraffin wax embedding procedures were used. Tissue samples from testicular parenchyma, pampiniform plexus and scrotal skin, were fixed in 10% buffered formaldehyde, dehydrated in a graded ethanol series, and cleared in xylene. Samples were embedded in paraffin wax and sections of 5 μm thickness were cut using a sliding microtome . Sections were stained with haematoxylin and eosin (H&E), periodic acid-Schifff (PAS) and Masson trichrome  and examined by light microscopy. Photomicrographs of each studied specimen were subjected to computer-assisted image analysis using a computer coupled to an optical Olympus BX50 microscope equipped with a Colour View IIIu digital Olympus DP27 camera .Parasite cysts found in the histological sections were observed in 10 randomized fields at a magnification of 100 (10 × 10) to obtain an average number of parasite cysts and to measure the diameter of the cysts.B. besnoiti bradyzoites [The immunohistochemical labelling of the parasites was performed using deparaffinised tissue sections from bulls with tissue cysts: bull 2 with numerous juvenile cysts; and bull 6 with scarce juvenile cysts and severe vascular lesions in the absence of cysts in the pampiniform venous plexus. Primary in-house rabbit polyclonal antibodies against dyzoites  were usedyzoites .A total of five bulls out of seven were analysed by different serological techniques. Four bulls showed high IgM values in the absence of specific IgG Table . The remBesnoitia spp. DNA was present in all scrotal skin samples (n = 7), followed by testicular parenchyma (n = 6) and the pampiniform plexus (n = 4). In total, 17 out of 21 samples analysed were positive (71%)  were detected in analysed tissues from three out of seven bulls  , with the exception of the pampiniform plexus of bull 6.Similar lesions were present in all tissues that included vascular lesions Fig. a–c.Fig. In bulls 1, 2, 4 and 7, diffuse thickening of the stratum corneum (hyperkeratosis) was observed in the epidermis; in one case (bull 2), nuclei were present in the keratinized cells (parakeratotic). An increase in the spiny layer (acanthosis) was also observed in bulls 1, 2, 4, and 7. We found areas with loss of epithelium (ulcers) and pustules in bull 6. In all cases, marked ecstasies of the sweat glands were evident Fig. f.Inflammatory infiltrate was predominant in all tissues. Some vessels had lymphocytic infiltrates in the wall (vasculitis). Hyalinosis and thickening of the basement membrane was also observed. Likewise, moderate hyperplasia of the smooth muscle fibres of the middle layer of both arterioles and venules was observed Fig. c, bull 2B. besnoiti naturally infected breeding bulls. We have demonstrated that a variety of severe acute lesions impair fertility in an early stage of the disease, as evidenced by different degrees of germline atrophy in the seminiferous tubules that are characteristic of an alteration in the development of the germ line and in a final step of testicular degeneration. Herein, a combination of serological, molecular and histopathological techniques was employed to characterize these case reports.The effect of acute besnoitiosis has been studied for the first time in seven All the sampled bulls showed clinical signs and/or macroscopic lesions compatible with acute bovine besnoitiosis mainly characterized by fever, orchitis and hydrocele. The laboratory diagnostic approach followed confirmed acute besnoitiosis in all bulls. However, the results showed that the infection progress differed among the sampled animals. Four bulls showed high IgM values in the absence of IgG . These results confirmed an acute infection within the first 2–3 weeks post-infection and prior to IgG seroconversion. Diezma-Díaz et al.  showed tThe presence of small tissue cysts 25–90 μm) in three bulls  also suggested a recent infection. Tissue cysts with a diameter between 48–67 μm were named juvenile tissue cysts by Basson et al.  since th0 μm in tB. besnoiti DNA burden was present in these tissues. This low parasitic load may be due to the initial replication of the tachyzoite in four bulls  and the detection of small cysts with a low number of bradyzoites inside [B. besnoiti DNA [7–1010 parasites/25 g of tissue (48–411 zoites/mg of tissue) [The parasite was able to spread through the organism, multiply and infect the target tissues of reproductive tract. McCully et al.  postulats inside  in the roiti DNA . However tissue) , 31, whe tissue) . The tac tissue) . In factIn our study, similar lesions were detected in the three testicular tissues analysed. A coexistence of lesions compatible with acute and chronic has been previously described in a naturally infected bull  and expeWe hypothesized that endothelial injury and inflammation are key pathogenic mechanisms responsible for testicular degeneration in acutely infected bulls Fig. . To the This study confirmed that severe acute besnoitiosis leads to early sterility that might be permanently supported by the severe lesions observed. The tachyzoite stage would trigger a cascade of lesions which worsen due to tissue cyst development. A rapid progression of the disease has been shown by the coexistence of characteristic lesions of both acute (vascular injury and inflammation) and chronic besnoitiosis (fibrosis and hyperkeratosis of the skin) that ends up with an alteration in testicle thermoregulation and the blood-testis barrier. However, markers of disease progression and prognosis remain to be clarified.