The transmission of viruses from animal hosts into humans have led to the emergence of several diseases. Usually these cross-species transmissions are blocked by host restriction factors, which are proteins that can block virus replication at a specific step. In the natural virus host, the restriction factor activity is usually suppressed by a viral antagonist protein, but this is not the case for restriction factors from an unnatural host. However, due to ongoing viral evolution, sometimes the viral antagonist can evolve to suppress restriction factors in a new host, enabling cross-species transmission. Here we examine the classical case of this paradigm by reviewing research on APOBEC3 restriction factors and how they can suppress human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). APOBEC3 enzymes are single-stranded DNA cytidine deaminases that can induce mutagenesis of proviral DNA by catalyzing the conversion of cytidine to promutagenic uridine on single-stranded viral (−)DNA if they escape the HIV/SIV antagonist protein, Vif. APOBEC3 degradation is induced by Vif through the proteasome pathway. SIV has been transmitted between Old World Monkeys and to hominids. Here we examine the adaptations that enabled such events and the ongoing impact of the APOBEC3-Vif interface on HIV in humans.  Spillover of viruses from animal hosts have led to the emergence of several human diseases including COVID-19, severe acute respiratory syndrome (SARS), Nipah, Ebola, influenza and acquired immunodeficiency syndrome (AIDS) ,3,4. SevThis positive selection of restriction factors leads to differences in functionality, even in similar species, resulting in the ability of these cellular restriction factors to also act as barriers to the cross species transmission of viruses . To succAIDS, which is caused by the human immunodeficiency virus (HIV), is a classic example of spillover of an animal virus into humans. HIV and related immunodeficiency viruses are lentiviruses, a genus of retroviruses where infection is characterized by long incubation periods between infection and onset of disease. The study of the emergence of AIDS also exemplifies how a cellular restriction factor can act as a cross species transmission barrier and how a virus adapts to overcome this barrier. This review examines cross species transmission of the simian immunodeficiency virus (SIV) between primates, with specific focus on the APOBEC3 family of cellular restriction factors that act as single-stranded (ss) DNA cytosine deaminases. The world first became aware of AIDS in the early 1980s, although it had become well established in the human population within the Belgian colonized region of the Congo in the early 1900s . Two lensmm of sooty mangabeys was transmitted to humans at least nine independent occasions giving rise to HIV-2 groups A-I . Aoccurred  115]. . cpz. Navif evolution than A3G, and thus is still important to consider in cross species transmissions .gor Vif interaction site for gorilla A3G also overlaps with HIV-1 Vif interaction site and is at position 16. However, for OWMs, their Vif interaction sites tended to cluster differently on Vif and were at Residue 84 for SMM and Residue 86 for RCM, although this is near Residues 15–17 (Several common interaction interfaces on both Vif and A3 enzymes have been identified. This is evolutionarily favorable since the interaction region can readily evolve without destabilizing the protein . Mutatioes 15–17 . The OWMes 15–17  151]. T. Tgor Vivif can become mutated to produce a protein that does not efficiently induce degradation of A3s [It has been suggested that inefficient interactions of Vif with A3s may actually be beneficial to the virus. During HIV-1 infection, n of A3s . This aln of A3s ,98,154. n of A3s ,155,156.n of A3s . Howevern of A3s ,159. ThuFuture work in the area of A3s and transmission barriers would be interesting to explore with other viruses. Recently A3B has been found to restrict certain herpesviruses and be antagonized by herpesvirus proteins . There mIn summary, A3 enzymes shaped the evolution of SIV and HIV through the ongoing suppression and avoidance related to Vif. As we learn of more viruses that are restricted by A3s in a deamination-dependent manner it would be interesting to realize if there are parallels with the SIV and HIV paradigm or new ones to be discovered. There is still much to learn about A3s and lentiviruses as we discover more A3 polymorphisms, ability of A3s to hetero-oligomerize, and the fate of A3-induced mutations in an organism over the lifetime of an infection.


Bulls frequently display male-male mounts, with consequences in the incidence of injuries, and possibly affecting the reproductive performance of the bulls. This behavior is known as the “buller syndrome” when appears in steers, with several individuals mounting one or a few penmates. The study aimed to collect information on the incidence of bull-bull mounts, the possible associated factors, the productive consequences, and management applied by distributing a survey to Holstein bull breeders in Uruguay. A survey was applied and responded by most Holstein breeders in Uruguay (30/33). Nineteen of the 30 breeders observed mounting behavior among bulls, and 15 of them considered it as a relevant problem. The breeders that observed the behavior had a greater number of bulls than those that did not observe it (P = 0.002). All of them observed that mounts were persistently directed towards the same individual . Of these, 11 (58%) considered that this stopped only when the “buller” bull was removed from the group, mentioning that the behavior was frequently redirected to another individual. The mounts between bulls are a major problem in the breeding of Holstein bulls, with important consequences on weight gain and animal health, reproductive problems such as low libido and seminal quality, and even provoking the death of animals. Although not all breeders reported the existence of the problem, those with the bigger herds did. While some management and/or environmental conditions seem to influence  the incidence of bull-bull mounts, there are no standardized managements to avoid this behavior. Considering that most breeders were interested in including practices to minimize this problem if available, it would be essential to understand better the causes and predisposing factors to decrease its negative impacts. Males of different mammal species, including most ruminants, frequently display homosexual behaviors, including male-male mounts  located in the south of the country: Canelones (3), Colonia (10), Flores (1), Florida (6), San José (7), and Soriano (3). At the time of the survey, these producers included a total of 265, 514, 90, 174, 155, 110 bulls in each of these Departments, respectively .The breeding system varied widely between breeders, with some of them raising based on natural pastures, others based in improved pastures, or paddocks with daily supplementation, with a density of 0.7 to 30 bulls/ha. Besides, in some cases the bulls were bred in the same paddocks with other categories, so the bull density was 0.05/ha.From the 30 breeders surveyed, 22 bred only Holstein bulls, and the other eight also bred Jersey (5), Angus (1), New Zealand variety (1), and Brown Swiss (1). Overall, 19 did not finish the animals with pen raising, while in the other 11, bulls were raised in pens from birth (3), since they were 4 months old (1), 9 months (1), 15 months (2) or only when the auction is approaching (1). Nine of the 30 breeders surveyed carried out their annual auction.The frequency of the visits of the breeder or the person in charge of the area where the bulls were varied widely, with two (10.5%) doing two daily visits, 11 (57.9%) one daily visit, 4 (21.1%) three visits/week, and two visits/week (10.5%). Several breeders had bulls in more than one paddock, so there were 8paddocks with less than five bulls, seven with 5-9, five with 10-19, six with 20-29, six with 30-50, and three paddocks with more than 50 bulls. Twenty-six of the 30 breeders (87%) regrouped bulls throughout the breeding period.Frequencies were compared with the Fishers’ exact probability test and the numerical variables with Anova.The article does not include the ethical approval for studies with animals because it does not include direct work with animals.Nineteen of the 30 breeders (63.3%) observed mounting behavior among bulls, and 15 of them (79.0%) considered it as a relevant problem. The size of the group was greater in those that observed the behavior than in those that did not observe it (P = 0.002), and the number of them that simultaneously had bulls from other breeds also tended to be greater (P = 0.076) . There wThe 19 breeders (100%) who reported the behavior observed that it was persistently directed towards the same individual . Of these, 11 (58%) considered that this stops only when the “buller” bull is removed from the group, mentioning that the behavior is frequently redirected to another individual. The “riders” bulls were “all the rest of the group” according to 13 breeders, those with the highest libido (eight breeders), the largest (seven breeders) or the most dominant (seven breeders), and those with the best body condition (three breeders) or the most aggressive (two breeders) (more than 100% as each breeder could respond more than one answer). Five breeders did not identify any particular characteristics. Twelve of the 19 breeders did not identify any special characteristic of the “buller” bulls. Others responded that it was the smallest of the group (nine responses), a new individual in the group (seven responses), the weakest (six responses), the one with the poorer body condition (two responses), that with bull-bull mounts aspect (two responses), or the least dominant individual (one response), a feminized individual (one response) or one that did not mate (one response).According to one breeder, the behavior is observed when the group has at least three animals; three breeders mentioned that it is observed when it has at least five animals, another that is when it has from seven to eight animals, 10 reported that it occurs when it has 10 to 15 animals per group, one that it is observed when the group has 15 to 20 bulls, two that the group should have 20 to 25 bulls, and one reported that it is observed in groups of more than 50 animals. Overall, 84% stated that they did not find a relationship between the size of the paddock and the incidence, but three breeders reported a higher incidence when the pasture is lower and the density higher.While 11/19 breeders (58%) reported that this behavior is more frequently observed in spring, the other eight found no link with the season of the year. No breeder reported links between the incidence of this behavior and the type of food. Nine of the 19 (47.4%) breeders reported that the incidence increases after regrouping animals, or moving and locking them up for sanitation or vaccinations, or selecting animals for sale. Only one breeder reported that he observed a greater number of mounts during the afternoon, without other responses related to daily variation.From the nine breeders who make their auctions, 4 (44.4%) reported that the mounts are more frequent during the handling for it. Two other breeders reported that both, when auctions are carried out and when semen is collected, the animals remain tied during the day, which influences an increase in the incidence of mounts between bulls when they are released at night.All the breeders that observed the behavior in their herds, observed injuries. Five breeders (26.3%) founded dead “buller” individuals. From all, 17 (89.5%) reported that the “buller” bulls had lower weight gains, 16 (84.2%) observed wounds on legs, 15 (80.0%) found the animals separated, in prolonged decubitus, and 14 (73.7%) animals with myiasis. Furthermore, 11 (57.9%) observed that the animals had shaggy hair, 7 (36.8%) abscesses in the rump region, three (15.8%) animals with diarrhea, and three other ischial peelings. Since some collect semen, four reported that sperm quality decreases. Besides, eight other different consequences were mentioned, including anus injuries, locomotor injuries such as hip dislocation, difficulties in moving, claudication, affections of the forelimbs, bruising, spinal cord injuries, general deterioration, and joint pain.Six breeders mentioned two specific leg injuries. One was described as “affection and removal of the upper phalanges of the hind limbs, and the second as “bladder in front of the hock” corresponding to synovitis/capsulitis of the tarsal joint. In addition, two breeders also mentioned the observation of penile fractures in “riders” bulls.In addition to these affectations, some breeders also reported on specific behaviors of the “buller” animal, including jumping wires and hiding in the trees, productive alterations such as failures in their pregnancies, low libido (does not mount), or that in many cases it was returned by the buyer.it is preferable to maintain the individual that is being harassed in the group under surveillance, withdrawing him only at imminent risk of death, than to removing him.” Two breeders decided to withdraw or not the “buller” bull depending on the situation, only when the injuries were serious. None of the breeders consulted removed the “rider” bull, explaining that in the number of animals/proportion it is easier to remove the few “bullers”.Eighteen breeders (94.7%) responded that they would be willing to take management measures if it is shown as useful to decrease the problems provoked by bull-bull mounts. The same number of breeders (18) took management measures when identifying the problem. Seventeen of the breeders that reported the occurrence (89.5%) removed the “buller” bull from the paddock, and the same number carried out specific treatments for the lesions . The only breeders that did not mention that they retire the “buller” bull responded “Eight breeders (42.1%) select older individuals when they are going to introduce new animals to a stable group, five (26.3%) declared taking other measures, including the confinement of all animals for 24 h before regrouping them  in a pen without food or drink, spraying them with creolin at the end of this period. Other measures mentioned to prevent mounts induced by regrouping were preadapting the animals during 4 days, maintaining them separated by an electric fence allowing their contact but without physical interaction; always raise them in subgroups separated by wire or in nearby pens so that they have already met each other; ensuring that the new ones to be introduced are more or less the same size (homogeneous) as the pre-existing group; maintaining small groups separated by categories (age/weight), and whenever possible, avoid regrouping and/or increasing the vigilance in the period following regrouping. In addition, three breeders (15.8%) mentioned that the size of the group to be introduced has to be between 30 and 50% of the pre-existing group size. Other breeders mentioned that the group to be introduced should have at least two, three, or four bulls (one mention each), at least five bulls, that has at least five bulls (mentioned by four breeders), seven to eight bulls (one mention), and at least 10 to 15 (four mentions). The other four breeders did not consider the size of the group of bulls to be introduced.To the best of our knowledge, this is the first report on the incidence of bull-bull mates studied, characterizing some factors that influence its incidence, its consequences, and possible strategies to minimize them. Although the behavior was not directly studied, the information was collected from Holstein bull breeders, covering more than 90% of the local breeders and most of the bulls marketed in the country, so it is possible to affirm that the data represents the view of the local Holstein bull breeders. This information is relevant to raise a problem that has not been systematically studied although it affects most bull breeders, but also farmers that raise non-castrated calves of this breed for fattening and slaughter, given that there is sustained information that they grow faster than castrated animals . Groups The information provided by the breeders confirms that there are several predisposing factors. The number of bulls in the group favors the display of the mounts between bulls, similarly as it was reported in steers . AlthougOne factor that was consistently mentioned as triggering this behavior is the regrouping of bulls. Grouping unknown animals produce an increase in aggressiveness with negative effects on their health and well-being . The groAlthough no breeder proposed it as management to reduce the incidence of the mounts between bulls, the number of breeders who reported its incidence was lower when they also bred together bulls from other breeds. Although according to our knowledge there is no specific information, there are differences in the incidence of behavior between bulls of different breeds, and some breeds may result more frequently in “bullers” roles . In consThe higher incidence in spring coincides with what was previously reported in steers . HoweverAlthough In summary, these data show that the mounts between bulls are a major problem in the breeding of Holstein bulls, with important consequences on weight gain and animal health, reproductive problems such as low libido and seminal quality, and even provoking the death of animals. Although not all breeders reported the existence of the problem, those with the bigger herds did it. While some management and/or environmental conditions seem to influence , there are no standardized managements to avoid this behavior, so it is essential to understand better the causes and predisposing factors to decrease its negative impacts. Moreover, most breeders were interested in including practices to minimize this problem if better tools are available. Most reported practices to reduce the incidence of this behavior are related to pregrouping practices, so it should be important to consider the experience of the breeders that could reduce this problem before regrouping bulls.


Lasius niger. After monitoring colonies over 64 weeks, body mass, neonicotinoid residues and virus titres of workers and queens, as well as worker behavioural activity were measured. ABPV, but not thiamethoxam, reduced activity of workers. Neonicotinoid exposure resulted in reduced body mass of workers, but not of queens. Further, thiamethoxam facilitated ABPV infections in queens, but not in workers. Overall, virus exposure did not compromise detoxification and body mass, but one colony showed high virus titres and worker mortality. Although the data suggest additive effects at the level of individuals and castes, co-exposure with both stressors elicited antagonistic effects on colony size. Our results create demand for long-term holistic risk assessment of individual stressors and their interactions to protect biodiversity.Pesticides and pathogens are known drivers of declines in global entomofauna. However, interactions between pesticides and viruses, which could range from antagonistic, over additive to synergistic, are poorly understood in ants. Here, we show that in ants the impact of single and combined pesticide and virus stressors can vary across castes and at the colony level. A fully-crossed laboratory assay was used to evaluate interactions between a sublethal dose of the neonicotinoid thiamethoxam and Acute bee paralysis virus (ABPV) in black garden ants,  This is of major concern because insects play indispensable roles in terrestrial ecosystems. They provide essential regulating and supporting ecosystem services of high economic value and crucial for food security, such as pollination, natural pest control, maintenance of soil fertility and soil formation5. Consequently, the reported declines are alarming and jeopardize the functions and services provided by insects7. The main drivers are habitat loss and fragmentation, anthropogenic pollution, invasive species, pathogens and climate change10. Furthermore, there is growing consensus that no single stressor is driving current declines, but rather complex interactions amongst a plethora of intertwined stressors12.The global entomofauna is affected by precipitous declines in both abundance and diversity13. The prophylactic abundant usage of non-specific pesticides to combat pest species inevitably harms non-target organisms15. As systemic pesticides, neonicotinoids are usually applied as seed coatings and only a minor fraction of the active ingredients is actually taken up by the plant roots, resulting in widespread environmental contaminations17. Even though environmental concentrations of neonicotinoids are often low, they can persist for a long time in the environment as they often have long half-life times (DT50)18. For example, the DT50 of thiamethoxam ranges between 7 and 92 days in field studies or between 34 and 233 days in laboratory studies21. For clothianidin, a metabolite of thiamethoxam22, the degradation is even slower with the field DT50 in the range of 13.3–305.4 days and DT90 ranging from 188 to more than 1000 days21. Chronic exposure of soil-dwelling organisms to fluctuating concentrations and mixtures of neonicotinoids in the range of 1 to > 100 ppb in arable farmlands are likely18. Therefore, they may induce long-term inadvertent sublethal effects on insect physiology and behaviour15. Harmful impacts on cognitive abilities, foraging behaviour, immune functions, colony development, fertility and reproductive output have already been reported to influence insects at individual, colony and community levels24. Of particular concern is a reduction in fitness, as this ultimately governs the state of all wild populations25. Social insects, which are to be considered among the most important insects from an ecological perspective26, are unsurprisingly also affected by exposure to neonicotinoid insecticides28.Today’s human food production profoundly relies on agro-chemicals, which represent one major anthropogenic pollution factor that acts as a known driver of insect declines26. These colonies are superorganisms, with individual insects being analogous to cells in multicellular organisms, acting as a functional unit30. Consequently, when studying the effects of stressors on eusocial insects, it is crucial to consider life-history traits specific to such superorganisms30. Therefore, a holistic understanding incorporates effects ranging from the individual, over the caste to the colony level, which in turn needs to be differentiated according to life-stages or caste-specific exposure risks and susceptibilities31. At an individual level, subtle effects of chronic exposure to sublethal insecticide doses can often be difficult to detect, yet over time they may translate into negative long-term consequences affecting a colony’s fitness32.Colonies of eusocial insects are characterized by overlapping generations, cooperative brood care and reproductive division of labour between castes, whereby one or a few female individuals (queens) reproduce, and the rest of the usually non-reproductive females (workers) perform all other tasks necessary to maintain the colony34. The combination of the long persistence of neonicotinoids21, perennial sedentary colonies and long life-spans, which characterize queens of many ant species, make long-term pesticide exposure highly likely35. Furthermore, long-term exposure inevitably leads to co-exposure and potential interactions with other stressors. Although interactions amongst stressors are often assumed to be synergistic per se, they may be additive or even antagonistic depending on the nature of the stressors and susceptibility of an organism37, which in turn underlies inter- and intra-species specific variability31. Factors such as age, developmental stage or sex influence stressor susceptibility leading to within-species differences38. While varying levels of susceptibility have been shown for haploid male (drone) and diploid female (worker) honey bees, data comparing queens and workers remain scarce31. In most species, queens and workers exhibit pronounced differences in morphology and longevity, and queens appear to be more tolerant of toxins39. Consequently, it seems plausible that susceptibility and stressor interactions vary between the two castes.Ants are ubiquitous social insects crucial for ecosystem functioning as they provide essential ecosystem engineering and services such as natural pest control and pollination41. Sublethal doses of pesticides may act as immune suppressors, thereby enhancing the spread and deleterious effects of pathogens44. For example, thiamethoxam and its metabolite clothianidin have been shown to affect the immunocompetence of honey bees, potentially enhancing susceptibility to viruses47. Viral agents affecting the health of Western honey bees (Apis mellifera) are well known48. However, with the rise of the invasive ectoparasitic mite Varroa destructor, the honey bee virus landscape has changed drastically50. This potent virus vector drives prevalence, titres and adaptions of viruses associated with it51. With elevated titres of these emerging infectious diseases in the apiary environment, the probability of host shifts increases52, ultimately affecting the wider insect community54. Viruses first described in honey bees, such as the two positive-sense single-stranded RNA viruses Deformed wing virus and Acute bee paralysis virus (ABPV), are now considered multi-host pathogens with a broad host range, including ants58. However, to our knowledge, no study has yet addressed the co-exposure to pesticides and viruses in ants41.Interactions between pesticides and pathogens have recently received considerable attentionLasius niger, are alternative hosts of ABPV, which can display clinical symptoms58. These common ants, endemic to Europe, can exploit all food resources known for ants except for direct granivory, and as ubiquitous generalist predators as well as being prey for other animals they play an important role in food webs59. Since they frequently scavenge on honey bees, exposure to honey bee viruses is likely and indeed there is a high prevalence of viruses in ants sampled in the surroundings of apiaries60. L. niger is characterized by medium to small-sized usually subterranean colonies in cities, gardens, meadows and arable lands59. Usually, the colonies are monogyne with extremely long-lived queens59 (up to 28 years61). Therefore, black garden ants represent an ideal model system to study potential interactions between long-term neonicotinoid insecticide exposure and viruses. We investigated sublethal effects of the neonicotinoid insecticide thiamethoxam and ABPV on L. niger at the colony and individual queen/worker level in a fully-crossed laboratory experiment. Thiamethoxam was chosen because it is amongst the most frequently used neonicotinoid insecticides62, and because it is metabolized into an active insecticide after entering the host, i.e. its primary metabolite clothianidin22. Over 64 weeks, newly founded colonies were chronically exposed to thiamethoxam (at a dose of 30 ppb64) or not (controls). Additionally, within each group, colonies were fed for 10 weeks with artificially ABPV infected honey bee pupae (A. mellifera) or control pupae. Workers were tested in a behavioural assay and we measured colony sizes, as well as neonicotinoid residue concentration, virus titres and body mass in both castes. Residue concentrations of both thiamethoxam and clothianidin were analysed to estimate levels of uptake and detoxification27.Here, we take advantage of recent findings that black garden ants, 65, and in ants a reduced movement speed and impaired locomotion have been described58. Further, ABPV infections can result in decreased colony size65. Sublethal effects of neonicotinoid insecticides on social insects include impaired locomotion, changes in activity, negative impacts on cognitive abilities, fertility, colony growth, reproductive output and effects on the immunocompetence66. Based on current literature, we therefore expected that both the neonicotinoid and the virus would affect the behaviour of workers and colony development and that thiamethoxam facilitates viral infections due to a compromised immune system, thereby resulting in a synergistic interaction of the two stressors.In bees, clinical symptoms induced by ABPV include quickly progressing paralysis trembling movements, inability to fly, loss of hairs on thorax and abdomen, gradual darkening appearance and adult mortalityX2 = 0.0, df = 3, p = 1.0). Overall, low worker mortality was observed, but it was not further traced in detail because dead ants were deposed on waste piles and therefore hardly recognisable67. However, at the end of the feeding regime (week 61), 37 workers were found lying dead in the foraging arena of one colony from the combined treatment. Six of the 40 queens were not able to raise workers successfully, i.e. not a single worker emerged, despite laying eggs , but neither of the two treatment factors (neonicotinoid—X2(1) = 0.78, p = 0.37, virus—X2(1) = 0, p = 1.0) nor the interaction term (virus:neonicotinoid—X2(1) = 0.0, p = 1.0) had a significant effect (N = 10 per treatment).Overall, queen mortality (i.e. colony failure) was 20% until the end of the experiment (week 64). There was no significant difference among the four treatments , virus = feeding regime with ABPV, combined = exposure to thiamethoxam and ABPV; each N = 10; log-rank test, F(1) = 16.7, p < 0.001), while neither the virus exposure nor the interaction term showed significant effects (virus—F(1) = 0.74, p = 0.4; interaction—F(1) = 0.71, p = 0.41; Fig. p < 0.01, Fig. F(1) = 0.4, p = 0.53; virus—F(1) = 0.91, p = 0.34; interaction—F(1) = 0.38, p = 0.54; Fig. The body mass of workers was significantly affected by neonicotinoid exposure  = 0.36, p = 0.83, Fig. X2(2) = 0.004, p = 0.95, Fig. F(1) = 20.4, p < 0.001, virus—F(1) = 7.7, p = 0.01, antagonistic interaction—F(1) = 4.8, p = 0.037; brood: neonicotinoid—F(1) = 21.9, p < 0.001, virus—F(1) = 6.6, p = 0.015, antagonistic interaction—F(1) = 7.0, p = 0.013; N = 8 per treatment). Post hoc pairwise comparison revealed that the controls had significantly more workers than the three treatment groups , which did not significantly differ among each other . Furthermore, controls had significantly more brood than the colonies from the neonicotinoid (p < 0.01) and the combined treatment (p < 0.01) , while the other treatment pairs were not significantly different amongst each other .Before the first overwintering (week 13), there were no significant differences in colony strength measured as the number of adults (F(1) = 7.3, p = 0.016) while neither the virus treatment nor the interaction term (caste:virus) showed significant effects (F(1) = 0.43, p = 0.52; F(1) = 3.48, p = 0.08; N = 8 per group; Fig. p-values > 0.23). A significant caste effect was determined for clothianidin residue concentrations (F(1) = 89.5, p < 0.01), while virus and the interaction term had no significant effect (F(1) = 0.25, p = 0.63; F(1) = 0.09, p = 0.76; N = 8 per group; Fig. p-values < 0.05), but there was no difference between queens or workers of the different treatments. Further, the queens had a significantly lower ratio of clothianidin to thiamethoxam ratio compared to workers (X2(1) = 17, p < 0.01).UHPLC-MS/MS analyses confirmed the absence of both thiamethoxam and clothianidin from all samples of the control and the virus treatment, except for one queen from the virus treatment Table . In the F = 7.3, pF = 7.3, pX2(1) = 32.9, p < 0.01), while thiamethoxam exposure and treatment interaction had no significant effect (neonicotinoid—X2(1) = 3.06, p = 0.08; interaction—X2(1) < 0.001, p < 0.99; N = 8 per treatment). No worker sample from the control treatment was tested positive for ABPV. In contrast, all samples of the virus and the combined treatment were positive, and interestingly two out of eight neonicotinoid treatment samples were positive (Table F(1) = 29.6, p < 0.01; neonicotinoid—F(1) = 0.02, p = 0.9; interaction—F(1) = 0.03, p = 0.86; Fig. Infection status (positive vs. negative) of workers were affected by virus exposure  = 13.74, p = 0.95; neonicotinoid—X2(1) = 4.66, p < 0.01) but no interactive effect was detected (X2(1) = 2.52, p = 0.11; N = 8 per treatment). The same holds true for virus titres in queens (virus—F(1) = 13.4, p < 0.01; neonicotinoid—F(1) = 6.42, p = 0.02; interaction—F(1) = 1.02, p = 0.86, additive effect; Fig. X2(1) = 0.42, p = 0.51), thus suggesting no significant caste effect.In queens, six out of eight were positive in the virus treatment, four out of eight in the neonicotinoid treatment, seven out of eight in the combined treatment and all controls were negative Table . The infX2 = 13.74,X2(1) = 0.94, p < 0.33, virus—X2(1) = 19.1, p < 0.01, interaction—X2(1) = 1.99, p = 0.16; N = 40 per treatment). Pairwise comparison showed that workers form the controls and the neonicotinoid treatment covered significantly more distance compared to the workers of the combined treatment  = 1.24, p = 0.27, virus—X2(1) = 0.57, p = 0.45, interaction—X2(1) = 1.5, p = 0.22; N = 40 per treatment; Fig. X2(1) = 0.46, p = 0.5, virus—X2(1) = 29.2, p < 0.01, interaction—X2(1) = 2.26, p = 0.13; N = 40 per treatment). The controls were significantly faster than the workers of the combined treatment (p = 0.002) and the workers of the neonicotinoid treatment were faster than the workers of the virus and the combined treatment , but no more significant pairwise differences were revealed by the post hoc testing  = 54.6, p < 0.01), while again, the factor neonicotinoid and the interaction term had no significant effects (X2(1) = 0.04, p = 0.84; X2(1) = 1.56, p = 0.21; N = 40 per treatment). Post hoc pairwise analysis revealed that workers of the control and the neonicotinoid treatment were significantly faster than the ones of the virus and the combined treatment . Although queen mortality and consequent colony failure are not affected, there is a clear effect at the colony level, where ABPV and thiamethoxam antagonistically affect colony strength. This implies that the colonies of the combined treatment do equally poor as the colonies exposed to individual stressors. Exposure to thiamethoxam appears to facilitate ABPV infections in queens, but not in workers. However, the exposure to virus seems not to affect the detoxification of the insecticide27, which matches the findings for imidacloprid68. Also, the queen’s ability to raise the first offspring was not affected. Even in the combined treatment, where one could expect increased stress, as the colonies are co-exposed to both stressors, no effects on either variable were detectable. Furthermore, we confirm that exposure to thiamethoxam reduces the body mass of workers, but not queens27. Body mass can be used as an indicator of health with potential implications on reproductive success70. Therefore, a reduction in body mass can be a symptom of an organism’s susceptibility towards a stressor71. In conclusion, the observed differences between queens and workers indicate that the latter are more susceptible towards thiamethoxam. In contrast, ABPV seems to have no effect on body mass irrespective of treatment or caste and no interactive effect with thiamethoxam was detected. However, exposure to ABPV resulted in impaired locomotion and reduced activity, confirming earlier results58: The distance covered, the average speed and the speed in the first seconds of observations were negatively affected by virus exposure. However, in contrast to our expectations, the behaviour of individuals was not affected by exposure to thiamethoxam. Moreover, no interactive effects of combined exposure were observed. In comparison, effects of neonicotinoid insecticide exposure on honey bee activity and locomotion have been shown, including longer inactivity, lower movement, and hyperactivity followed by hypoactivity72. Many aspects like foraging and competition (intra- and interspecific) are restricted by impaired locomotor abilities. For example, a reduced movement speed increases the time required to exploit available resources, which in turn can affect the colony growth rate73. A reduced work force impairs colony functioning, reduces competitive strength and decreases the number of produced sexuals77. Therefore, it is possible that the effects of virus exposure on locomotion can result in a reduction of colony performance and ultimately fitness .Across all treatments, we observed low mortality of queens (i.e. colony failure) and workers. We can confirm that thiamethoxam at 30 ppb appears to have only sublethal effects on ants78. However, there was one exception, as the workers found dead in one colony towards the end of the experiment reached more than 107 copies per mg tissue. Why titres were higher in these specimens and why worker mortality was induced in this particular colony, but not in others, remains speculative. Our results suggest that ABPV infections can be lethal in L. niger, in addition to symptoms reported earlier58. A comparison of honey bees and ants is meaningful in this regard because these viruses have been studied in depth in the former48, but not in the latter. Both ants and honey bees are highly eusocial with reproductive division of labour, overlapping generations and cooperative brood care and therefore, they share key aspects of their life-history, which are clearly more important than other biological traits such as body size33. Indeed, comparisons of honey bees with solitary and even primitively eusocial bees (e.g. bumblebees) are not as appropriate despite obvious similarities in the pollinator guild79, due to superorganism resilience and other key features of eusociality30.To determine possible caste specific differences in susceptibility towards ABPV, we used virus titres as an indication of infection levels. When normalized to body mass, there were no differences regarding the virus load of queens and workers. Despite the detection of clinical symptoms (i.e. impaired locomotion) the observed virus titres did not reach the levels of overt infections in honey bees43. Clothianidin, the metabolite of thiamethoxam, modulates immune signalling, thus reduces immune defences and promotes virus replication in honey bees bearing covert infections45. However, the effect of thiamethoxam on the susceptibility towards ABPV were observed exclusively in queens, but not in workers. As the life expectancy of queens is much higher compared to workers, the former might invest more into detoxification, which in turn could negatively affect the immune response towards viruses. Another possible explanation could be that neonicotinoid insecticides interfere with the social structure of colonies or their ability to detect a pathogen, restricting social immune defences and thus increasing the chances of ABPV to reach the queen80. If or how neonicotinoids may affect the interaction network and social immunity in ants, as it has been suggested for bumblebees32, remains to be investigated. Some samples of the control and the neonicotinoid treatment being tested positive for the virus can perhaps be attributed to feeding with untreated honey bee pupae, which may have been infected by this virus. Indeed, although magnitudes lower than in artificially infected pupae, low background levels of viruses (i.e. covert infections) commonly occur in field-collected honey bee pupae65.In queens, the data indicate that chronic exposure to thiamethoxam can result in higher virus titres. This suggests that the immune system may be weakened and the susceptibility towards ABPV increased, thereby supporting the idea that insect diseases can be linked to pesticides27). This could also explain that thiamethoxam affects the body mass of workers but not of queens. Just as neonicotinoids facilitate viral infections45, we might expect an inverted effect where the induced stress of an ongoing virus infection vice versa affects detoxification negatively. However, exposure to ABPV had no detectable effect on the residue concentration of either thiamethoxam or clothianidin. This would be in line with findings in honey bees, where co-exposure to thiamethoxam and chronic bee paralysis virus resulted in an increase in virus loads, but neither the survival nor the ability to metabolize the insecticide to clothianidin was affected46. Noteworthy is the observation that one queen of a treatment group without thiamethoxam was tested positive for the insecticide. Given that no negative control was positive, thereby excluding cross-contaminations, the most parsimonious explanation is exposure in the field prior to sampling.Further differences between the castes were revealed when looking at neonicotinoid residues to confirm uptake and metabolization of thiamethoxam. The residue levels indicate that queens seem to be able to cope better with the intoxication, indicating superior detoxification  of the combined treatment was not different from the neonicotinoid and virus treatment. Thus, at colony level, the two stressors interact antagonistically, meaning that co-exposed colonies are doing better than expected if the individual effects of viruses and neonicotinoids are summed up81. These findings are in line with studies in bees suggesting that interactions between pesticides and pathogens are often antagonistic, particularly interactive effects on proxies of fitness such as behaviour, parasite load and immune responses37. Overall, these results highlight that the nature of stressor impacts and interactions strongly depends on the variable and effect level looked at. Examples such as the increase in susceptibility of queens towards ABPV when exposed to thiamethoxam demonstrate that effects at an individual level do not necessarily translate to colony level effects. When studying stressor impacts on social insects, it is thus crucial to consider colony-level effects. Superorganism resilience and social immunity are just two factors which show that effects on individuals have limited explanatory power when it comes to colony fitness30. A limitation of this study is that colony fitness was not assessed directly, as L. niger typically does not produce sexuals within the first years of colony development33. Therefore, we had to rely on colony size as a token of fitness in L. niger. Nonetheless, the observed effects are likely to affect colony fitness, because the number of workers and impaired locomotion affect many factors of colony functionality75, especially foraging as well as intra- and interspecies competition76. Lastly, the onset of the reproductive stage is earlier and more sexuals are produced in colonies with a larger workforce77. In this experiment, only a single concentration of thiamethoxam was tested, which is not sufficient for the demonstration of a dose–response or more complex response patterns such as dose-dependent synergism/antagonism83. Therefore, our results are only valid at the used neonicotinoid concentration and further experiments with a range of varying thiamethoxam concentrations would be required to draw definite conclusions about combined effects with ABPV.Our data revealed that the effect of thiamethoxam exposure on colony strength only becomes visible in the second year of colony development and that in colonies exposed to virus fewer adults emerged84. Hence, future studies should address how frequently and how long ants are exposed to stressors under field conditions and measure how high residues are in such specimen. Furthermore, the relative importance of multiple simultaneous exposure routes (acute and chronic) such as contact with spray droplets, contaminated soils, foliage, and water, as well as consumption of contaminated nectar, pollen, guttation fluid and seeds, honeydew, invertebrate prey and water are all poorly understood28. The exposure scenario in this laboratory study only covers exposure via contact with contaminated cotton reflecting contaminated soil and consumption of contaminated water reflecting soil pore water, neglecting additional exposure pathways. Uptake via consumption of water seems to be more important than topical contact with the toxicant because of the weak hydrophobicity of the neonicotinoids yielding a low penetration through the cuticula66. Another topic not addressed within the scope of this experiment is the question of bioavailability, i.e. what fractions of neonicotinoids present in soils are available for uptake, and for causing adverse effects to biota85. Therefore, the extent of the actual exposure of ants in contaminated soils remains elusive. Nonetheless, the neonicotinoid levels used in this experiment can be considered as a worst-case scenario as they were constantly in the upper range of what can be found in arable fields18. Furthermore, it can be assumed that such arable fields are probably less suitable habitats for ants in the first place because frequent disturbance, e.g. ploughing, will interfere with the construction of nest structures for perennial sedentary colonies. Nevertheless, ants frequently settle in arable lands86, and even if newly mated queens are successful in establishing a colony at the edge of such fields, long-term chronic exposure at lower dosages is likely17.The use of chronic exposure in the laboratory constitutes a conservative approach as there are no foraging risks due to predation, no competition and additional pathogens or unfavourable weather conditions. Furthermore, field colonies are usually exposed to multiple pesticides simultaneously, and exposure peaks that can occur during times of insecticide applications are absent in such a laboratory setting89. Moreover, long-term effects as well as combined exposure scenarios with multiple pesticides and/or other stressors are mostly neglected so far90. Therefore, we emphasize the need to fully incorporate long-term effects, combined exposure scenarios and ants as model organisms in holistic future risk assessments. We argue that incorporating fitness as a key endpoint variable in future pesticide risk assessment schemes may substantially help to ensure more reliable and robust stressor evaluations25. This would subsequently mean that for eusocial insects the lifetime reproductive output of sexuals should be assessed either directly or with a reliable proxy in multi-generation laboratory essays to mitigate the future impacts of chemicals and other stressors on biodiversity25.In conclusion, our results show that a holistic view is required to understand how an organism is affected by interacting stressors. Life-history traits, all levels of action, acute and long-term effects need to be considered as well as co-exposure to multiple stressors. In our case, it would not have been possible to estimate colony-level effects based on individual variables or if measurements were taken too early. Furthermore, effects could vary over time and therefore it would be necessary to monitor ant colonies over their entire life-cycle to understand the ultimate impact of any stressor. However, in current pesticide risk assessment schemes none of the indicator species for soil-dwelling insects are comparable to ants and other social insectsL. niger colonies allocated to four treatment groups. The four treatment groups were (i) controls, (ii) virus—colonies fed with honey bee pupae artificially spiked with ABPV, (iii) neonicotinoid—colonies chronically exposed to thiamethoxam (30 ppb), and (iv) combined—colonies co-exposed to thiamethoxam and ABPV. No ethical approval was required to work with these invertebrate species and all experiments were performed in accordance with relevant guidelines and regulations. Gynes were collected in the field after their nuptial flights , the colonies were transferred into new nesting tubes with an attached foraging arena  directly dissolved in distilled water. To test for the effects of chronic thiamethoxam exposure over the full experimental duration (64 weeks), a concentration of thiamethoxam was chosen (30 ppb) as it may be detected in soils of arable farmlands64. This concentration is below the level found in fields where the insecticides have just been applied but it is in the range of what can be found in fields up to 1 year post treatment18. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to confirm neonicotinoid persistence in pooled treatment solutions collected from the nesting tubes at the moment of nest translocation (week 34) and at the end of the experiment (week 64)27.The nesting tubes were separated by a sterile cotton wool ball into two compartments. The front compartment, closed by another cotton wool plug, housed the colony while the hind chamber was filled with 10 ml treatment solution59. Post hibernation, colonies received a sugar-water (40% mass fraction of sugar) drenched cotton ball and four fruit flies (Drosophila hidey) weekly. With the start of the treatment feeding regime, we switched from Drosophila to Western honey bee (Apis mellifera) pupae. The colonies in the virus treatments received honey bee pupae artificially spiked with ABPV, while the controls received control pupae for 10 weeks. For this, white-eyed honey bee pupae were obtained from sealed worker brood frames of local colonies . Using standard methods91, half of the pupae were microinjected laterally between the second and third segment of the abdomen with 2 μL of an ABPV solution for virus propagation. Then, all pupae were incubated in darkness at 34.5 °C, ≥ 50% RH for 5 days. Virus presence in injected pupae was confirmed using quantitative real-time polymerase chain reactions  and pupae were frozen at − 80 °C until needed.Before the first overwintering, colonies were provided twice with 30 μl droplets of sugar-water (50% mass fraction of sugar) once the first workers emerged, due to claustral colony foundingThe amount of brood  and the number of workers were assessed before the first overwintering (week 13) and at the end of the experiment after freezing the colonies (week 64). Further, the numbers of workers and pupae were counted at the start of the second treatment (week 51). The body mass of individual queens and twenty pooled workers was measured using an analytical balance  at the end of the experiment.58, 2 weeks after the feeding regime ended (week 63). In brief, the movement of individual ants in an arena (Θ = 90 mm) was video recorded for 120 s. Motion data were visually evaluated from slow motions recordings by quantifying the movement of ants in relation to a reference grid (grid cell: 8.5 mm × 8.5 mm), i.e. the number of grid cells passed over time. Only one grid cell was counted if an ant touched two cells at the same time, e.g. when walking along a line. Thereby, the overall movement, time inactive, average speed and initial speed was assessed. Overall movement was defined as the number of grid cells passed during the entire 120 s, time inactive as the time the ants did not move (including grooming and standing still), average speed as the number of cells passed in the time the ants were actively moving and initial speed as the number of cells crossed during the first 10 s. For more information and an exemplary video see Schläppi et al.58.For each colony, the behaviour of five randomly chosen foragers sampled from the foraging arenas was analysed, as described in detail in Schläppi et al.27. In brief, an adapted QuEChERS protocol was used to prepare and purify pooled samples with worker abdomens and individual abdomens of queens. Subsequently, an Acquity UPLC system  coupled to a TQ-S triple quadrupole (Waters) were used for the analyses of the purified samples. Solutions in MeOH 25% at 0.005, 0.05, 0.5, 5 and 15 ng/ml, each containing internal standards (concentration of 5 ng/mL) were used for internal calibration to quantify thiamethoxam and clothianidin followed by linear regressions weighted by 1/x. We then normalised the samples to their mass and expressed them as ng/g dry mass for comparisons. The quantification detection limits were 20 pg/g of tissue for thiamethoxam and 40 pg/g for clothianidin. We included blank samples as negative controls.Queens and workers of all colonies that lasted until the end of the experiment  were analysed for both thiamethoxam and its metabolite clothianidin using UHPLC-MS/MS analysis as depicted in Schläppi et al.92. However, only the head, thorax and extremities were used, as the abdomens were used for the neonicotinoid analyses. Each sample was manually crushed with a mortar in TN buffer . Fifty microlitres of the homogenate were then used for the RNA extraction with a NucleoSpin RNA II kit . 0.3 ng Ambion RNA Control 250 was added to each sample at the first extraction step to monitor the efficiency of the RNA purification and cDNA synthesis93. After elution in 30 µL of elution buffer, RNA was stored at − 80 °C. Then, a M-MLV RT Kit  was used for reverse transcription by incubating the template RNA (0.05–0.25 μg) in a Thermocycler  with random hexamer oligonucleotide  and RNase-free water in a final volume of 17.75 μL for 5 min at 70 °C. For, the cDNA synthesis, 5 × Buffer (5 μL), nucleoside triphosphate  and reverse transcriptase  were added to a final reaction volume of 25 μL and incubated it at 37 °C for 60 min. The obtained cDNA was diluted 1/5 and used for RT-qPCR to estimate ABPV titres using a KAPA SYBR FAST Universal qPCR kit . A duplicate of each sample was run for both the targeted virus and the exogenous internal reference in a 12 μL reaction mix containing SYBR green reaction mix (6 μL), Milli-Q water (2.52 μL), diluted cDNA (3 μL) and each 0.24 μL of the forward and reverse primers . In addition, four ten-folds serial dilutions (10−2 to 10−5 ng) of purified PCR products used as standard curves and two no-template negatives were included on each plate. The reaction was processed in an ECO Real-Time PCR machine  according to the following cycling profile: 95 °C for 3 min (incubation) followed by 40 cycles of 95 °C for 3 s (denaturation) and 57 °C for 30 s . Fluorescence reading at 0.5 °C intervals between 55 °C and 95 °C was used to perform a melting curve analysis after the amplification for the verification of the specificity of the PCR products. RT-qPCR output data, standard curves and the experimental dilution factors were used to calculate an estimate of viral copies per sample (virus titres). To account the exponential distribution virus titres and for body mass differences between the castes, virus titres were log-transformed, normalized to the mass of the samples and expressed as “Log10 genomic copies / g dry mass” throughout the manuscript and for the statistical analyses. A sample was considered negative with zero viral copies if it had a shifted peak or no peak at all in the melting curve analysis. For both castes, Cq values of the negative samples were transformed to hypothetical virus titres and of the maximum value of these “negative titres” a titre detection threshold was obtained normalized to the castes mean bodyweight.First, RNA was extracted from the queens and a pooled sample of 40 workers from each colony using standard methods95. The Shapiro–Wilk test and the Levene’s test were used to test data and model residues for normal distribution and homogeneity of variances for the selection of statistical tests. Colonies were used as individual replicates unless specified differently and upon queen death, the respective colony was no longer included in further analyses. Consequently, sample sizes decrease from N = 10 per treatment upon initiation of the experiment to N = 8 per treatment at the end.R version 3.6.3 was used to run all statistical analyses and create Figs. survdiff function from the R package survival96. The ability of queens to raise workers (binary response variable), i.e. if queens were able to lay eggs and attend eggs/larvae/pupae adequately so that at least one viable worker emerges (success or failure), was analysed using a generalized linear model with binomial distribution97. Both treatments  and their interaction term (cross-product term—virus:neonicotinoid) entered the model as fixed effects. Queen and worker body mass were analysed using linear models with body mass of either caste entering the model as a response variable again with both treatments and their interaction as fixed effects. ANOVA was used as a likelihood ratio test to obtain p-values by comparing the model with the effect in question against the model without it. If a model revealed significant main effects, it was followed up by pairwise comparisons using Tukey’s Honest Significant Difference test, thereby controlling for multiple comparisons. Based on the definitions of an additive effects model, synergism or antagonism occur, when combined effect of multiple stressors is greater (synergism) or less than (antagonism) the sum of effects elicited by the individual stressors82. Therefore, if the interaction term is non-significant additive effects were assumed and else, if the interaction (cross-product term) of the two stressors was significant, a deviation from additive effects was assumed. In these cases, we consulted the model estimates to claim synergism  or antagonism 82.Queen survival was analysed with a log-rank test using the art function from the R package ARTool98. For cross-factor pairwise comparisons, we followed the developer’s recommendations and used Mann–Whitney U tests on the original data for each treatment pair and then corrected the results for multiple comparisons using Holm's sequential Bonferroni procedure correction.Colony strength, measured as the number of workers and the amount of brood  was analysed before the first overwintering and at the end of the experiment. Because of the nature of the data and as only the thiamethoxam treatment was established before the first overwintering, colony strength was compared between two groups  using a Kruskal–Wallis test (non-parametric). The number of workers at the end of the experiment entered a linear model as response variable and was analysed with both treatments and their interaction as fixed effects. As above, post hoc pairwise comparison was performed using Tukey’s Honest Significant Difference test. The amount of brood (response variable) was analysed using an aligned rank transformation for nonparametric factorial analyses with same fixed effects using the lmer function of the R package lme499. Colony identity was used as a random factor because samples of the same colony are not completely independent97.Infection status of queens and workers each were analysed using a generalized linear model with binomial distribution and virus titres were analysed with linear models as described above. To meet the model assumptions for workers the response variable virus titre was log-transformed. Furthermore, the relationship between virus titre (response variable) and ant caste (fixed effect) was analysed with treatments and colony identity as random factors using a linear mixed effect model with the 92. All samples of the control and virus treatment were negative except for one queen sample, which was positive for thiamethoxam and clothianidin. Non-positive negative controls confirmed that there was no detectable contamination. Therefore, only the samples from the neonicotinoid treatment and the combined treatment were used in the subsequent analysis. Consequently, each substance (concentration per gram dry mass) entered the model as a response variable together with caste and virus treatment as fixed effects and colony identity as a random factor to respect that both the queen and workers of each colony were tested. For pairwise comparisons, we then used Wilcoxon signed-rank tests respecting when queens and workers of the same colonies were compared (paired = TRUE). The ratio of clothianidin to thiamethoxam was calculated for all samples positive for both substances . Then, the ratio was log-transformed and analysed using a linear mixed-effect model with caste as a fixed effect and treatment as well as colony identity as random factors.To compare neonicotinoid residues (thiamethoxam or clothianidin) detected in queens and workers among the treatments we again used aligned rank transformation for nonparametric factorial analyses91. In all cases, both treatments and their interaction were used as fixed effects with recording date and colony identity as random factors to control for potential effects of the recording day and measurements of five workers per colony that should not be regarded as independent measurements.The variables of the behavioural assay overall movement and average speed were analysed with linear mixed effect models, while time inactive and initial speed each entered a Poisson linear mixed effect model due to the nature of their data, which reflects counts with large values being rare events, i.e. Poisson distribution48. In the second publication, the long-term impact of chronic neonicotinoid exposure on ant colony development was investigated22, based on the two neonicotinoid groups reported in this manuscript. Here, although we confirm the findings of the previous studies with the expanded dataset, we focus on the interaction of the two factors thiamethoxam and ABPV, to provide an independent contribution to the ongoing debate about stressor interactions. Due to the special focus on stressor interactions, the overlap is mostly restricted to the material and methods section.Parts of the data reported in this manuscript have been previously published in two other manuscripts. The first publication focused on foodborne transmission and clinical symptoms of honey bee viruses in ants and contained, among other data, the two neonicotinoid control groups reported in this manuscriptSupplementary Information.


Sexually transmitted infections (STIs) and HIV can generate costs both within and outside the health sector . This systematic review aims (i) to explore the intersectoral costs associated with STIs and HIV considered in cost-of-illness (COI) studies, (ii) to categorise and analyse these costs according to cost sectors, and (iii) to illustrate the impact of intersectoral costs on the total cost burden.Medline (PubMed), EMBASE (Ovid), Web of Science, CINAHL, PsycINFO, EconLit and NHS EED were searched between 2009 and 2019. Key search terms included terms for cost-of-illness, cost analysis and all terms for STIs including specific infections. Studies were included that assessed intersectoral costs. A standardised data extraction form was adopted. A cost component table was established based on pre-defined sector-specific classification schemes. Cost results for intersectoral costs were recorded. The quality of studies was assessed using a modified version of the CHEC-list.Patient & family, Informal care and Productivity (Paid Labour). Patient & family costs were addressed in four studies, including patient out-of-pocket payments/co-payments and travel costs. Informal care costs including unpaid (home) care support by family/friends and other caregiver costs were considered in three studies. All six studies estimated productivity costs for paid labour including costs in terms of absenteeism, disability, cease-to-work, presenteeism and premature death. Intersectoral costs largely contributed to the total economic cost burden of STIs and HIV. The quality assessment revealed methodological differences.75 COI studies were considered for title/abstract screening. Only six studies were available in full-text and eligible for data extraction and narrative synthesis. Intersectoral costs were captured in the following sectors: It is evident that intersectoral costs associated with STIs and HIV are substantial. If relevant intersectoral costs are not included in cost analyses the total cost burden of STIs and HIV to society is severely underestimated. Therefore, intersectoral costs need to be addressed in order to ensure the total economic burden of STIs and HIV on society is assessed, and communicated to policy/decision-makers.The online version contains supplementary material available at 10.1186/s12913-021-07147-z. Th. Th27]. The search strategy, as part of a larger systematic literature review, identified 21,935 articles after de-duplication. Due to the high number of records identified between 1999 and 2019, studies were further limited to 2009-2019, which led to the exclusion of 6,426 studies. This time period was selected to reflect the greater attention focused on incorporating intersectoral costs in economic analyses over the past ten years , 29, 30.Table Patient & family, Informal care and Productivity (Paid labour) [Table  labour) –35. One Patient & family costs were addressed in four studies, with some costs being related to healthcare services and treatment within a national insurance system and others to patient-borne expenses outside an insurance system , 33–35. Two studies explicitly reported to have captured informal care costs, which were concerned with unpaid (home) care support by family or friends , 35. MosProductivity costs can involve productivity losses for paid labour and non-paid opportunity costs . All six studies estimated patients’ productivity costs for paid labour, including costs in terms of absenteeism, short-term/partial/long-term disability, cease-to-work, presenteeism or premature death , 31–35. The impact of HIV-related productivity costs per year per patient on the overall costs were presented in three studies and varied between 9 % and 41 % , 34, 35,The quality of the COI studies did not vary considerably but methodological differences were evident . Five stThis review is the first to explore whether existing COI studies carried out for STIs and HIV considered intersectoral costs in their analyses, and to categorise these according to specific cost sectors. Further, it clearly demonstrates that intersectoral costs significantly contribute to the total cost burden of STIs and HIV.Only a small number of COI studies were identified that captured intersectoral costs of STIs and HIV. This small number implies that intersectoral costs are often overlooked in the literature and remain largely excluded from COI studies in this area. Some might argue this finding could indicate that intersectoral costs are not relevant, but this review concludes differently. For example, this review provides evidence that productivity losses for people living with HIV can account for up to 40 % of the total costs per year.There are several reasons that could explain why many studies ignored the wider scope of costs, even though intersectoral costs for STIs and HIV can be substantial . One reaThis review suggests that the COI studies that adopted a societal perspective tend to predominantly assess healthcare and productivity costs related to paid labour. This is in line with previous research reporting that even when a societal perspective is adopted in economic studies these often only consider healthcare and labour costs . The focThe present findings suggest that the assessment of unpaid labour and non-paid opportunity costs such as leisure time, volunteering and care for children or elderly is rather limited in COI studies. Similar findings were found for full economic evaluations  , howeverAs mentioned earlier, intersectoral costs can have a big impact on the total cost burden. In fact, this review reveals that the inclusion of intersectoral costs attributed to STIs and HIV indicate a substantially higher cost burden to society than healthcare costs alone. This means that unless intersectoral costs are taken into account, the total cost implications of STIs will not be appreciated. This is in keeping with a review of COI studies by Pike and colleagues (2015) which reported that limiting the assessment of the economic burden to healthcare costs can substantially underestimate the total economic cost burden .This review found that the heterogeneity of methodological approaches in COI studies, including the choice of study perspective(s) and what costs to include in analysis, made it rather difficult to analyse and compare the impact of intersectoral costs across studies. The use of different methods in cost analyses can affect how results are interpreted and subsequently affect policy decisions. This review raises awareness on the potential need for standardised guidelines for COI studies and a standard quality assessment tool for COI studies to assess the consistency and transparency of these studies and improve comparability.The present review shows that only a small number of COI studies of STIs and HIV include intersectoral costs. Those studies that do capture intersectoral costs tend to report a higher burden for STIs and HIV, which is important information for policy/decision-makers. These findings imply that if intersectoral costs are not included in cost analyses, the total cost burden of STIs and HIV to society is severely underestimated. If intersectoral costs are captured in COI studies this may change the overall results and is likely to improve the information developed for decision/policy-makers. Realising the higher cost burden of STIs and HIV might give more prioritisation to interventions targeted at reducing the number of STIs and HIV compared with other competing demands on the healthcare budget.The strength of this review lies in its rigorous and systematic approach. A comprehensive search strategy was developed in collaboration with an information specialist. Studies were carefully screened to evaluate whether intersectoral costs had been captured and a cost component table (or classification scheme) was established that can be adapted or expanded by future research, as needed. The present review considered studies conducted in all OECD member countries to account for a good representation of study results. Further, this review was able to synthesise evidence that addresses the impact intersectoral costs of STIs and HIV can have on the total economic burden. This review also has some limitations. One limitation is that other studies that potentially assessed intersectoral costs may have been missed. The articles were limited to the timeframe of 2009-2019. Five potentially relevant articles eligible for full-text analysis were only available in form of abstracts or posters. After finding that only six studies were eligible for data extraction and narrative synthesis and available in full the review team scanned a random number of excluded studies to check whether potentially relevant articles may have been missed. Further, the complex nature of STIs and HIV requires an examination of the wider societal impacts, and the included studies might not represent the fuller range of potentially relevant cost sectors. This however also means that the present classification scheme could serve as a guide for future research and offers room for expansion.Overall, this review has generated pertinent evidence and presents a clear message that the focus of most of the existing COI studies to date is largely on healthcare costs when it is evident that the impact of disease is wider and more substantial.Future research could further investigate relevant cost sectors associated with STIs and HIV and validate or complement the findings of this review. Gathering more evidence could help propose a standardisation of cost classifications for COI studies concerned with STIs and HIV. Economic evaluations could be reviewed to identify the different sector-specific costs as well as benefits associated with interventions targeting STIs and HIV.It is evident that intersectoral costs associated with STIs and HIV are substantial and largely contribute to the total economic cost burden. However, studies tend to predominantly assess healthcare and productivity costs related to paid labour under a societal perspective. If relevant intersectoral costs are not included in cost analyses the total cost burden of STIs and HIV to society is severely underestimated. Therefore, intersectoral costs associated with STIs and HIV need to be addressed in order to ensure the total economic burden of STIs and HIV on society is assessed, and communicated to policy/decision-makers.Additional file 1Additional file 2Additional file 3


N-isopropylacrylamide-co-n-butyl methacrylate) hydrogel was applied to beads of various pore sizes: 7, 12, and 30 nm. Almost the same amount of copolymer hydrogel was applied to all beads, indicating that the efficiency of copolymer modification was independent of pore size. Analyte retention on prepared beads in a packed column was observed using steroids, benzodiazepines, and barbiturates as analytes. Analyte retention times increased with temperature on packed columns of 12- and 30-nm beads, whereas the column packed with 7-nm beads exhibited decreased retention times with increasing temperature. The difference in analyte elution behavior among the various pore sizes was attributed to analyte diffusion into the bead pores. These results demonstrate that bead pore diameter determines temperature-dependent elution behavior.Temperature-responsive chromatography using thermoresponsive polymers is innovative and can control analyte retention via column temperature. Analyte elution behavior in this type of chromatography depends on the modified thermoresponsive polymer and the structure of the base materials. In the present study, we examine the effect of the pore diameter of silica beads on analyte elution behavior in temperature-responsive chromatography. Poly( A variety of stationary phases for liquid chromatography have been developed for various applications. One of them is using thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), modified stationary phase for temperature-responsive chromatography7. PNIPAAm exhibits thermoresponsive hydrophilic and hydrophobic property changes that can be attributed to hydration and dehydration in response to temperature change9. Since the phase transition temperature of PNIPAAm (32 °C) is close to the human body temperature, it has relevance to various biomedical applications such as drug and gene delivery systems14, biosensors and bioimaging systems19, temperature-modulated cell separation systems23, and cell culture substrates for tissue engineering27. In a chromatography system, PNIPAAm-modified silica beads are used as the stationary phase, and hydrophobic interaction between PNIPAAm and the analyte is modulated by changing temperature (via temperature-responsive PNIPAAm hydrophobicity changes). The chromatography system uses all-aqueous mobile phases, although the addition of a small amount of methanol is required to improve detection with mass spectroscopy28. To improve the performance of chromatography systems, various types of PNIPAAm copolymer have been investigated such as the copolymerization of hydrophobic monomers to enhance hydrophobic interaction32 and the copolymerization of ionic monomers to utilize electrostatic interaction besides hydrophobic interaction37. Moreover, several types of PNIPAAm configuration on silica bead surfaces have been investigated using various polymerization methods40. Therefore, almost all studies on temperature-responsive chromatography focus the PNIPAAm on silica beads surface. However, although the structure of silica beads, especially pore diameter, is an essential factor in the separation efficiency of a chromatographic stationary phase, this has not been studied in temperature-responsive chromatography.Liquid chromatography is one of the most effective separation analysis methods across several biomedical fieldsco-n-butyl methacrylate (BMA)), a PNIPAAm copolymer with a hydrophobic co-monomer, hydrogel was used to modify the silica bead surfaces. The elution behavior of the packed column of prepared beads was assessed using steroids, benzodiazepines, and barbiturates as model analytes because these molecules have different chemical structures and hydrophobicities for temperature-responsive chromatography. Moreover, these analytes represent different types of drugs: steroids are anti-inflammatory drugs, whereas benzodiazepines and barbiturates are psychoactive drugs. Thus, the separation of these analytes would exhibit the versatility of drug analysis using temperature-responsive chromatography. Analyte diffusion into the pores was investigated using pullulan with a range of molecular weights. The effect of the pore diameter of the temperature-responsive stationary phase on the elution behavior of the analytes has been investigated.In the present study, to investigate the effect of pore diameter on analyte elution in temperature-responsive chromatography, stationary phases were prepared using silica beads with various pore diameters  as base materials  copolymer hydrogel  before and after modification by the P(NIPAAm-co-BMA) copolymer was successfully modified through this reaction. The estimated amount of grafted polymer was almost the same among all beads. This result indicates that the efficiency of hydrogel modification on the silica bead surfaces was independent of the pore diameter of the silica beads.The prepared beads were characterized by elemental analysis Table . The car42. Relatively larger surface area and pore volume were observed in the 12-nm pore-diameter aminopropyl silica beads compared to those of 7- and 30-nm pore diameter, indicating that the 12-nm diameter beads had greater porosity compared to the other beads. This was probably due to the production method for 12-nm aminopropyl silica beads being slightly different than that for other pore-diameter beads. Pore diameter, pore volume, and surface area slightly decreased after polymerization because the polymer hydrogel modified the inside of the bead pores. However, the results indicated that the porous structure remained in all beads after copolymer hydrogel modification, i.e., the bead pores did not become clogged.To investigate the porous property of the beads before and after polymer hydrogel modification, the pore diameter, pore volume, and surface area of the beads were investigated by nitrogen adsorption and using the Brunauer–Emmett–Teller (BET) method Table 41,42. ReThe prepared polymer hydrogel-modified beads were packed into a stainless-steel column . The elution behavior of various analytes from the bead-packed column was observed.Five hydrophobic steroids with similar molecular weights and various hydrophobicities were used as analytes Table . The temP. This result indicates that steroid retention occurred via hydrophobic interaction between the polymer and analytes. Packed columns of beads with 12- and 30-nm pore diameters exhibited an increase in the retention time of the hydrophobic steroids with increasing column temperature, and effective separation was observed at the highest temperature of 45 °C. For example, dexamethasone and hydrocortisone acetate were eluted as an overlapped peak at 15 °C. With increasing temperature, dexamethasone and hydrocortisone acetate were separated and eluted as two separate peaks. This is because the enhanced hydrophobic interaction between copolymer hydrogel on the silica beads and the hydrophobic steroids could be attributed to the dehydration of the copolymer on the bead surfaces at a higher temperature. By contrast, beads packed column with 7-nm pore-diameter beads exhibited decreasing analyte retention times with increasing column temperature. The differences in the retention profiles were likely attributable to the pore diameter of the base beads.On all columns, the retention time of the steroids increased with increasing analyte hydrophobicity, LogFor all the columns, the peak widths decreased with increasing temperature. This is attributed to the temperature-dependent shrinkage of the thermoresponsive polymer hydrogel layer on the silica bead surface. At low temperatures, this polymer layer was swollen, and the analyte tended to diffuse into it. Thus, relatively broad peaks were observed at low column temperatures. Contrastingly, the silica bead polymer layers shrunk at high temperatures, and the analyte did not tend to diffuse into the hydrogel layer, leading to relatively sharp peaks at high temperatures.By comparing peak shapes among the various pore diameter sizes at high temperatures, sharp peaks were observed for the small pore-diameter-bead packed column compared to the large pore-diameter-bead packed column. This is probably attributable to the difference in the tendency of the analyte to diffuse into the pores. For large pore-diameter beads, the analyte tended to diffuse into the pores and interact with the polymer hydrogel layer inside the silica beads, leading to broad peaks.44. Large increases in the retention time of steroids were observed in the lower temperature regions. This is because of the phase transition temperature of the copolymer used on the beads. A similar P(NIPAAm-co-BMA) copolymer hydrogel exhibited phase transition at approximately 20 °C21. Thus, modified hydrogel dehydration likely occurred in this temperature region, leading to the retention of the steroids through enhanced hydrophobic interaction. By contrast, on the 7-nm pore-diameter beads, retention time decreased with increasing column temperature  exhibited low-resolution separation at low temperatures. Thus, the developed small pore-diameter beads are useful for the separation of an analyte at low temperatures, which is advantageous for the separation of analytes that tend to deactivate and decompose at relatively high temperatures.These results demonstrate that a column using small pore-diameter beads (7 nm) can control analyte retention via changes in column temperature in a manner opposite to that previously reported for temperature-responsive chromatography columnsTo further investigate the elution profiles of analytes from columns packed with beads of various pore diameters, the elution of benzodiazepines from the column was observed Figs. . BecauseFurthermore, the elution behavior of the barbiturates was observed as additional analytes Figs.  because co-BMA) hydrogel inside the pores shrunk, leading to pore size enlargement in the beads and enhanced diffusion of the analyte into the pores. An even larger increase in elution volume with temperature was observed for the columns using large pore-diameter beads. This is because the analyte was more likely to diffuse into large pores than into small pores at high temperatures. Elemental analysis of copolymer hydrogel-modified beads revealed that a similar amount of copolymer was present on the bead surfaces  exhibited inverse temperature-dependent analyte retention profiles compared with those with relatively large pore diameters (12 and 30 nm). The difference in elution profiles was attributed to analyte diffusion into the pores.The changes in retention profiles during temperature-responsive chromatography were affected by three factors, i.e., (1) hydrophobic interaction, (2) analyte diffusion into pores, and (3) analyte solubility in the mobile phase. (1) The enhancement of the hydrophobic interaction between the polymer and analyte was attributed to the dehydration of the polymer with increasing temperature, which led to an increase in retention time with temperature. (2) Additionally, analyte diffusion into the pores was enhanced with increasing temperature, leading to an enlarged effective area for hydrophobic interaction between the polymer and analyte. (3) By contrast, analyte solubility in the mobile phase increased with increasing temperature, leading to a decrease in analyte retention. Analyte retention during temperature-responsive chromatography resulted from a combination of these factors. In the case of the 12- and 30-nm pore-diameter beads, these three factors affected analyte retention. With increasing temperature, increased hydrophobic interaction between the copolymer and analyte was attributed to the dehydration of the copolymer and an increase in the effective surface area, leading to an increase in analyte retention. By contrast, for the 7-nm pore-diameter beads, the diffusion of the analyte into the pores was relatively suppressed compared with that for the larger-pore-diameter beads. Hence, with the 7-nm pore-diameter beads, the effective surface area for hydrophobic interaction did not increase with increasing temperature. By contrast, analyte solubility into the mobile phase increased with increasing temperature, leading to a decreased retention of the analyte.The results indicate that bead pore diameter influences the retention behavior of analytes in temperature-responsive chromatography. Using relatively large pore diameter (12 and 30 nm) beads as packing materials, the retention time of an analyte can increase with increasing temperature. By contrast, when using small pore diameter (7 nm) beads, retention time can decrease with increasing temperature, which has not been observed in temperature-responsive chromatography. The small pore-diameter-bead packed column achieved high-resolution separation at low temperatures, whereas the relatively large pore-diameter-bead packed columns (12 and 30 nm) exhibited low-resolution separation at high temperatures. Thus, the development of small pore-diameter-bead packed columns would be useful for the separation of analytes at low temperatures, which has advantages for the separation of analytes that tend to deactivate and decompose at relatively high temperatures.The effect of pore diameter of base silica beads on analyte retention in temperature-responsive chromatography was investigated using silica beads with pore sizes of 7, 12, and 30 nm. Elemental analysis indicated that almost the same amount of copolymer hydrogel was applied to the beads independent of bead pore size. Nitrogen adsorption analysis revealed that the pores of the silica beads after hydrogel modification did not become clogged, although pore diameter slightly decreased. The elution behavior of analytes from bead-packed columns was observed using steroids, benzodiazepines, and barbiturates as analytes. Differences in temperature-dependent elution profiles were observed between the column packed with 7-nm pore-diameter beads and those packed with 12- and 30-nm pore-diameter beads. For the columns packed with 12- and 30-nm beads, the retention time of the analytes increased with increasing temperature, whereas retention time decreased with increasing temperature for the columns packed with 7-nm pore-diameter beads. To investigate analyte diffusion into the bead pores, the temperature-dependent elution volume of pullulan was observed. The elution volume of pullulan indicated that its diffusion into pores was enhanced with increasing temperature for the 12- and 30-nm pore-diameter beads owing to the shrinkage of the polymer hydrogel inside the pores. By contrast, for the 7-nm pore-diameter beads, the enhancement of analyte diffusion into the pores was relatively small compared with that for the 12- and 30-nm pore-diameter beads. These results suggest that small pore-diameter beads can obtain the high-resolution separation of analytes at low temperatures, whereas large pore-diameter beads can separate analytes at high temperatures. Thus, the development of small pore-diameter beads would be useful for the separation of analytes at low temperatures, which has advantages for the separation of analytes that tend to deactivate and decompose at relatively high temperatures.co-BMA) hydrogel-modified silica beads with various pore diameters were prepared by the following procedure  were dispersed in the solution. The bead dispersion was degassed by N2 gas bubbling in an ultrasonic bath for 30 min. Then, the flask was sealed, and the V-501 immobilization reaction was proceeded with continuous shaking for 17 h at 25 °C. After the reaction, V-501 modified beads were filtered and rinsed with ethanol. The beads were dried at 25 °C in vacuo.P, and N,N′-methylenebisacrylamide  were dissolved in 50 mL of ethanol in a 100 mL flask. Then, V-501 immobilized silica beads (2.0 g) were dispersed in the reaction solution and the solution was degassed by N2 gas bubbling in an ultrasonic bath for 30 min. Next, polymerization was proceeded for 15 h at 70 °C. After polymerization, the beads were filtered and rinsed using methanol. The beads were dried in vacuo, and P(NIPAAm-co-BMA) hydrogel-modified beads were obtained.NIPAAm , The prepared silica beads were characterized by CHN elemental analysis, FE-SEM observation, and nitrogen adsorption.CI is the increase in carbon composition after V-501 immobilization reaction, %CI  is the calculated carbon percentage of V-501, and S is the surface area of the beads.The carbon composition of the beads was obtained via CHN elemental analysis . The amount of immobilized V-501 on the silica beads was obtained using the following equation:Cp is the increase in the carbon composition of the copolymer hydrogel-modified beads relative to that of the V-501 modified beads and %CP  is the calculated percentage of carbon in the polymer hydrogel.The amount of modified thermoresponsive hydrogel was obtained as follows:The bead morphologies of aminopropyl group modified beads and polymer hydrogel-modified beads were observed via FE-SEM .42.Pore diameter, pore volume, and surface area were measured by nitrogen adsorption . These parameters were calculated using the BET methodPrepared polymer hydrogel-modified beads were packed into stainless-steel columns (4.6 mm diameter × 100 mm length). Beads were suspended in a methanol/water (1:1) solution. Then, the suspension was poured into a column packer connected to the column. Bead packing was performed by flowing the methanol/water solution at a constant pressure of 350 kg/cm for 1 h. The bead-packed column was connected to an HPLC system . The elution behavior of analytes from the column was observed using steroids Table , benzodik’ was obtained using the following equation:tR is the analyte retention time and t0 is the retention time of uracil as an initial standard45.To investigate analyte retention behavior on the column, Van’t Hoff plots for the analytes were obtained. The retention factor Additionally, the elution of glucose and pullulan standard samples from the columns was observed to investigate the molecular weight of the analytes and the pore size of the beads. Pullulan was detected using an RI detector .Supplementary Information.


Reproductive systems of cattle contain multiple microbes resident in the female from a young age. Sometimes other harmful microbes can invade the genital tract and cause diseases that impair fertility. Normally, commensal microbes facilitate genital tract homeostasis and produce factors that stimulate male sexual response. For this reason, the type and number of microbes present in the genital tract are important for reproductive tract health, and any disruption of this microbial balance leads to genital diseases. Interestingly, these microbes frequently populate the genital tract of cows, leading to reproductive diseases that perturb fertility. However, a microbiome composed of commensal microbes will likely result in the restoration of uterine health and improved fertility of the cows.Cattle have a genital microbiome that is established early in life, even before calving. Microbial influx into the reproductive system of cows, during calving or mating, is unavoidable and is likely to alter the commensal microflora composition. It is now well established that a commensal endometrial flora is largely responsible for the overall fertility of cows. These microbes are important for maintenance of structural integrity of the genital mucosal barrier, immunomodulation, and protection against pathogens. Further, the genital microbiome functions in the semiochemical communication between a male and female. An optimal balance between the abundance and diversity of the microbiome is essential to promote female genital tract health. Disruption of this balance leads to dysbiosis and genital diseases and perturbed fertility. As part of the global strategy of One World, One Health, there is a need to reduce antibiotic use in animals. This area of research has the potential to expand the knowledge about the nexus between the endometrial microbiome and fertility including being probiotic in different species. Bacteroidetes and Fusobacteria and lower relative abundance of Proteobacteria and Tenericutes . Vag. Vag8]. In humans, alteration of normal microbial diversity can lead to dysbiosis, infertility  or genitThe traditional dogma is that the endometrial environment is sterile, particularly during pregnancy . It is nBacteroidetes, Fusobacteria, and Proteobacteria are the most common phyla in the genital tract . Res. ResBacteriaceae .8%, in aabundant . In the F. necrophorum, Porphyromonas Levii, and T. pyogenes were detected in pregnant cows [Histophilus and Mycoplasmataceae, can become pathogenic [It is now known that cows have a natural microbiome in the uterus during the gestational period ,59. Whilant cows . Interesthogenic ,62. Furtthogenic .Lactobacilli use their small membrane extensions  that adhere to the genital mucosa [Aggregatibacter spp. [The genital microbiome changes during the lifetime of females. Microbial populations in the reproductive tracts of animals are naturally selected because of different symbiotic functions. For example, in women, l mucosa . Likewister spp. ,64. Therter spp. . The thoter spp. . Howeverter spp. . When thIndividual variation in microbial species in the genital tract of bovids is thought to have effects of fertility outcomes . This vaEnterococcus spp., Staphylococcus spp., and Streptococcus spp., which was different from the vaginal microbiome of ewes where there was a predominance of Bacillus spp., Corynebacterium spp., Escherichia spp., Staphylococcus spp., and Streptococcus spp.  (MRSA) skin infections (64–82%) compared to when neonates are born without complications  [The type of fetal delivery at parturition is one of the major factors affecting the genital microbiome diversity . The genelivery) .Bacteroidete spp., Histophilus somni, Actinobacillus seminist, and unclassified Fusobacterium populations increase when there are relatively lower estradiol concentrations in the vaginal milieu  gene that modulates degranulation and phagocytic processes [L. rhamnosus, Pediococcus acidilactici, and L. reuteri resulted in a marked reduction of the inflammatory profile of endometrial epithelial cells when E. coli was administered or included in the culture medium [S. aureus, one of the major pathogens implicated in postpartum infections in dairy cattle, was inhibited in vitro under treatment with L. gasseri CRL1421 and CRL1412 [In cattle, intravaginal treatment with a mixture of lactic acid bacteria, e by 58% . The prorocesses ,82,83. Trocesses ,84. Resue medium . Likewis CRL1412 .The genital microbiota represents an opportunistic field of study in the realm of cattle fertility when considering the microbiome is an essential immunological barrier against pathogens and for pheromone production. Specific questions that can be addressed are the functions of specific bacteria taxa involved in physiological uterine involution in postpartum cows. With dysbiosis, uterine disease can occur; thus, modulating the vaginal microbiome may be an effective alternative to antibiotic therapies. Therefore, intravaginal inoculation of cattle at risk of infection represents possible probiotic management of genital postpartum uterine infections.