Production of offspring from cloned transgenic RFP female dogs and stable generational transmission of the RFP gene

The purpose of this study was to analyze the reproductive ability of transgenic female dogs born bysomatic cell nuclear transfer and to determine inheritance of the red fluorescent protein (RFP) transgene. The four founder transgenic bitches (F0) reached puberty at 340.8 ± 39.6 days after birth and were bred with wild-type male dogs by natural mating or by artificial insemination. The bitches all became pregnant and successfully delivered 13 puppies (F1), of which two females were bred with wild-type dogs to deliver 7 offspring (F2), including 1 stillbirth. Among the 19 live offspring, 10 puppies showed emission of RFP under UV light and the presence of the RFP transgene was confirmed by genomic PCR and Southern blot analyses. In conclusion, transgenic RFP female dogs exhibited normal reproductive ability and expression of the transgene was demonstrated in F1 and F2 generations.     

Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer

To improve the efficiency of somatic cell nuclear transfer (SCNT) in dogs, we evaluated whether or not the interval between fusion and activation affects the success rate of SCNT. Oocytes retrieved from outbred dogs were reconstructed with adult somatic cells from a male or female Golden Retriever. In total, 151 and 225 reconstructed oocytes were transferred to 9 and 14 naturally synchronized surrogates for male and female donor cells, respectively. Chromosomal morphology was evaluated in 12 oocytes held for an interval of 2 hr between fusion and activation and 14 oocytes held for an interval of 4 hr. Three hundred seventy-six and 288 embryos were transferred to 23 and 16 surrogates for the 2 and 4 hr interval groups, respectively. Both the male (two pregnant surrogates gave birth to three puppies) and female (one pregnant surrogate gave birth to one puppy) donor cells gave birth to live puppies (P > 0.05). In the 2 hr group, significantly more reconstructed oocytes showed condensed, metaphase-like chromosomes compared to the 4 hr group (P < 0.05). A significantly higher pregnancy rate and a greater number of live born puppies were observed in the 2 hr group (13.0% and 1.1%, respectively) compared to the 4 hr group (0%) (P < 0.05). In total, three surrogate dogs carried pregnancies to term and four puppies were born. These results demonstrate that decreasing the interval between fusion and activation increases the success rate of clone production and pregnancy. These results may increase the overall efficiency of SCNT in the canine family.     

A cloned toy poodle produced from somatic cells derived from an aged female dog

To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.     

Anti-sperm antibodies and seminal characteristics after testicular biopsy or epididymal aspiration in dogs

A study was performed to determine if performing testicular biopsies or epididymal aspirates in dogs would induce sperm-bound anti-sperm antibodies (ASA), affect long-term sperm production or semen quality. Semen was collected from 8 mature dogs 3 times a week before and after hemicastration and then 3 times a week after testicular biopsy (n=3 and 1 control) or epididymal aspiration (n=3 and 1 control). Detection of anti-sperm IgG (ASA) on sperm cells was performed by flow cytometry analysis using a flow cytometer. Two dogs with testicular biopsies became positive for ASA 16 d after testicular biopsy and remained positive for 7 and 9 d, respectively. One dog that had an epididymal aspirate became positive 13 d after epididymal aspiration and remained positive for 35 d. One dog became positive 21 d after hemicastration and remained positive for 28 d. Sperm output declined significantly in 7 of 8 dogs after hemicastration. A control epididymal aspirate treatment dog had decreased sperm output, and a testicular biopsy treatment dog had increased sperm output. None of the dogs with ASA had significant changes in sperm output after treatment. Sperm motility declined significantly in 3 dogs after hemicastration. An epididymal aspiration treatment dog had a decrease in sperm motility, a control epididymal aspirate treatment dog and a control testicular biopsy treatment dog each had increases in sperm motility. None of the dogs with ASA had significant changes in motility. The percentage of normal spermatozoa significantly decreased in 3 dogs and significantly increased in 1 dog after hemicastration. Two dogs that had testicular biopsies and 1 dog that had an epididymal aspiration had decreases in percent normal sperm. Two of 3 dogs with decreases in percent normal sperm after treatment had ASA, but 2 dogs with ASA had no change in motility. Hemicastration, epididymal aspiration, and testicular biopsy can induce ASA production within 2 wk of the procedure, but ASA are transient and do not have a predictably negative effect on total sperm output or motility. Testicular biopsy and epididymal aspiration are safe diagnostic procedures, but further work investigating post-treatment fertility must be done before final conclusions can be made.     

Reproductive fertility of cloned male cats derived from adult somatic cell nuclear transfer

This study was designed to investigate the reproductive fertility by the natural breeding of cloned male cats with domestic female cats, and to measure endocrine hormone concentration related to male reproduction such as testosterone, leutinizing hormone (LH), and follicle stimulating hormone (FSH). Cloned A, B, C, and D cats produced three, two, four, and five kittens after natural mating with four domestic female cats, respectively, despite later puberty of the cloned D cat than those of the other cloned male cats. Three of the 14 kittens expressed an odd eye color, which was produced by 1 and 2 from cloned A and B cats. The eye color of the other F1 kittens varied from nine brown to two blue. Body weight at birth ranged from 72.9 to 134.0 g. Although clone D had a poorer libido and entered puberty later than those of the other cloned male cats, he produced gonadal hormones within the average range. Four of the cloned male cats had normal fertility. The concentration of gonadal hormones in cloned male cats was similar to two control and donor cats. The concentration of testosterone was not significantly different among clones A, B, C, D, and control cats (5.99 +/- 5.68; 3.46 +/- 2.81; 6.41 +/- 2.17; 3.75 +/- 0.34; 4.0 +/- 3.63 ng/mL, p < 0.05). The concentrations of LH and FSH were not significantly different among the cloned cats (p < 0.05). Seven male and seven female (in total 14) kittens were produced by the natural breeding with four domestic female cats. These results indicated that cloned male cats have normal reproductive fertility and lie within the normal range of gonadal hormone production. All F1 kittens were produced by natural breeding and delivery, and are still alive and have normal growth health (27 months age).     

Influence of female reproductive anatomy on the outcome of sperm competition in Drosophila melanogaster

Females as well as males can influence the outcome of sperm competition, and may do so through the anatomy of their reproductive tracts. Female Drosophila melanogaster store sperm in two morphologically distinct organs: a single seminal receptacle and, normally, two spermathecae. These organs have different temporal roles in sperm storage. To examine the association between sperm storage organ morphology and sperm competition, we used a mutant type of female with three spermathecae. Although the common measure of sperm competition, P(2), did not differ between females with two and three spermathecae, the pattern of sperm use over time indicated that female morphology did affect male reproductive success. The rate of offspring production by females with three spermathecae rose and fell more rapidly than by females with two spermathecae. If females remate or die before using up second male sperm, then second male reproductive success will be higher when they mate with females with three spermathecae. The results indicate that temporal patterns of sperm use as well as P(2) should be taken into account when measuring the outcome of sperm competition.     

The evolution of sperm length in moths

Sperm form and function remain poorly understood despite being of fundamental biological importance. An instructive approach has been to examine evolutionary associations across comparable taxa between sperm characters and other, potentially selective reproductive traits. We adopt this approach here in a comparative study examining how sperm lengths are associated with male and female reproductive characters across moths. Primary data have revealed Lepidoptera to be an ideal order for examination: there is profound variation in the dimensions (but not organization) of the reproductive traits between closely related species which all share a monophyletic ancestry, for example, eupyrene sperm length varies from 110 to 12,675 microm. Eupyrene (normal fertilizing) and apyrene (anucleate and non-fertile) sperm lengths are positively correlated across taxa and both sperm types show positive associations with mating pattern (as measured by the residual testis size). At fertilization, eupyrene sperm must migrate down the often elongated female spermathecal duct from storage to unite with the ovum. Across taxa, the elongation of this duct is associated with increased eupyrene sperm length, suggesting a positive female influence on sperm size since longer, more powerful sperm may be selected to migrate and/or compete successfully down greater ductal lengths. Apyrene sperm length is not associated with female reproductive tract dimensions. However, we found a positive relationship between the residual testis volume and spermathecal volume, suggesting coevolution between male investment in spermatogenesis and the extent of the female sperm storage capacity. Within males, there is a positive association between the two organs which form the ejaculate-containing spermatophore: the testes and the accessory gland. The 'trade-up' in investment to these components is discussed in relation to paternal investment and mating patterns.     

The knobbed acrosome defect in four closely related dogs

The knobbed acrosome defect (KAD) has only once been reported in dog sperm. This report describes the breeding history, clinical presentation and semen evaluation of four closely related miniature Schnauzer dogs that had between 8 and 44% sperm with KAD. Two of the dogs had no substantial reproductive or sperm defects other than the KAD and were of normal fertility.     

Birth of viable female dogs produced by somatic cell nuclear transfer

Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6-22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.     

Development of play behavior between potential guide dogs for the blind and human raisers

This study shows longitudinal development of human-dog interactions in a guide dog raising program. Social play interactions between potential guide dogs for the blind (puppies) and their adult female raisers (PWs) were videotaped at home during the period from two months to 11 or 12 months of the puppies' age. The puppies and PWs established close proximity relationships by two to three months of age. The older the puppies became, the shorter time the puppies and PWs spent in intensive fighting and chasing play. The older the puppies became, the longer the puppies and PWs spent in waiting, seeking and possessing together play which needed the puppies' self-control, concentration and ability to cooperate with the PWs. Whether or not these behavioral tendencies which the puppies showed were appropriate for guide dog candidates is also discussed.     

Artificial insemination in canids: a useful tool in breeding and conservation

Artificial insemination (AI) and semen freezing have become services available to dog owners worldwide, and the demand for services to freeze semen is increasing. In other canids such as the fox, the fur industry utilizes fresh or frozen semen to artificially inseminate vixens to produce pelts. Clearly, AI facilitates the use of a male to sire several females by diluting the ejaculate, increases breeding hygiene, and allows crossing between species with slightly different breeding seasons. The African wild dog (Lycaon pictus) is currently considered by the World Conservation Union (IUCN) as one of most endangered canids. In captive populations of African wild dogs, semen has been frozen with encouraging results, using a standard cryopreservation protocol for domestic dogs, but successful AI has not been reported. In wolves, there is one report regarding the live birth of an offspring after intravaginal AI of a deslorelin-induced estrous female. In 2005, three Mexican gray wolf females were artificially bred by intrauterine insemination with freshly collected semen from unrelated males, and all females whelped. Artificial insemination may be vaginal, intrauterine or intratubal, and the semen may be fresh, fresh and chilled (diluted), or frozen-thawed, and the source of semen may be epididymal or ejaculated. In the domestic dog, the results are good to excellent for AI with all three types of processed semen when the source is ejaculated semen, whereas epididymal sperm still yields poorer results. Species differences in female physiology, as well as differences in the cryotolerance of the sperm from various canid species, warrant further research and development.     

Semen characteristics of genetically identical male cats cloned via somatic cell nucleus transfer

We investigated the sperm characteristics of four cloned male cats (Felis catus) to assess their reproductive potential. Fresh and frozen-thawed sperm were assessed for motility, viability, and morphology, and their functional competence was evaluated by in vitro fertilization (IVF) of domestic cat oocytes. All fresh semen characteristics varied among cats and collection times. Sperm concentration (× 10⁶/mL) of Cat A (512 ± 140, range 368 to 685) was significantly higher, whereas that of Cat C (335 ± 92, range 274 to 469) was significantly lower than that of Cloned B (459 ± 159, range 336 to 510) and control cats (680 ± 452, range 360 to 479). After thawing, motility and progressive motility of sperm from Cat B were significantly lower than that of the other cloned and control cats. The curvilinear, straight line, and average path velocities of sperm from Cat B were significantly higher, whereas the straightness was lower, than that of the other cloned and control cats. Frozen sperm from Cats A, B, and C successfully fertilized oocytes (cleavage = 74.4%, 71.4%, and 86.2%, respectively) and produced embryos that developed to the blastocyst stage after IVF/In vitro culture (IVC) (34.4%, 26.7%, and 48.0%) at frequencies similar to the cleavage rate (82.0%) and blastocyst rate (43.9%) obtained with sperm from the control male. In conclusion, seminal characteristics of cloned male cats did not differ markedly from those of our noncloned, control male cats.     

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.     

Female behaviour and the interaction of male and female genital traits mediate sperm transfer during mating

Natural selection and post‐copulatory sexual selection, including sexual conflict, contribute to genital diversification. Fundamental first steps in understanding how these processes shape the evolution of specific genital traits are to determine their function experimentally and to understand the interactions between female and male genitalia during copulation. Our experimental manipulations of male and female genitalia in red‐sided garter snakes (Thamnophis sirtalis parietalis) reveal that copulation duration and copulatory plug deposition, as well as total and oviductal/vaginal sperm counts, are influenced by the interaction between male and female genital traits and female behaviour during copulation. By mating females with anesthetized cloacae to males with spine‐ablated hemipenes using a fully factorial design, we identified significant female–male copulatory trait interactions and found that females prevent sperm from entering their oviducts by contracting their vaginal pouch. Furthermore, these muscular contractions limit copulatory plug size, whereas the basal spine of the male hemipene aids in sperm and plug transfer. Our results are consistent with a role of sexual conflict in mating interactions and highlight the evolutionary importance of female resistance to reproductive outcomes.     

Molecular characterization of trichomonads from feces of dogs with diarrhea

Trichomonads are occasionally observed in the feces of dogs with diarrhea. On the basis of superficial morphological appearance, these infections have been attributed to opportunistic overgrowth of the commensal, Pentatrichomonas hominis. However, molecular characterization of canine trichomonads has never been reported. This study was performed to determine, by means of rRNA gene sequence analysis, the identity of trichomonads observed in feces from dogs with diarrhea. Total DNA was isolated from fecal samples obtained from a 3-mo-old mixed breed dog and litter of German Shepherd puppies having profuse liquid diarrhea containing numerous trichomonads. Total DNA was subject to PCR amplification of partial 18S rRNA gene or 5.8S, ITS1, ITS2, and partial 18S and 28S rRNA genes using species-specific and universal primers, respectively. Products of 642 and 1864 base-pair length were amplified and cloned. On the basis of rRNA gene sequence, the trichomonads observed in the single dog and the litter of puppies shared 100% identity with Tritrichomonas foetus and P. hominis, respectively. The present study is the first to establish the molecular identity of trichomonads infecting dogs with diarrhea. These studies validate the longstanding assumption that canine trichomoniasis may be attributed to P. hominis. Importantly, these studies additionally recognize that canine trichomoniasis may also be caused by infection with T. foetus.     

Ejaculate production schedule and the degree of protandry in fishflies (Megaloptera: Corydalidae)

1. To examine the relationship between male–female emergence patterns and ejaculate dynamics, patterns producing sperm and seminal fluids in male internal reproductive organs, the size of a spermatophore transferred at mating and the fate of spermatophore contents moved into female storage organs were compared among the fishflies (Megaloptera: Corydalidae: Chauliodinae), Parachauliodes continentalis, P. japonicus and Neochauliodes sinensis .
2. Spermatophore contents moved into female storage organs decreased rapidly in P. japonicus and N. sinensis , but hardly at all in P. continentalis . This suggests that the females of the former two species may remate sooner than the latter species as it is known in insects that material remaining in the storage organs mechanically inhibits receptivity to mating.
3. Male P. japonicus and N. sinensis increased in internal reproductive organ mass continuously after adult eclosion, and the spermatophore size produced at the first mating increased with male age. In contrast, the internal reproductive organs of P. continentalis were relatively small and did not increase in mass after emergence. P. continentalis transferred a constantly smaller spermatophore at any copulation than the former two species.
4. Males of P. japonicus and N. sinensis emerged earlier than females, while P. continentalis showed a nearly simultaneous emergence pattern between the sexes. It seems that males of P. japonicus and N. sinensis (more polyandrous than P. continentalis ) inhibit female receptivity for a longer time period by emerging earlier and transferring larger ejaculates. In these two species, the number of sperm ejaculated at the first mating also increased with male age. The increase in sperm number by emerging earlier may be adaptive for the males owing to numerical sperm competition when the female remates.     

Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer

As shown by the birth of the first cloned dog ‘Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.     

Sperm Quantity Explains Age-Related Variation in Fertilization Success in the Hide Beetle

Age-related variation in sperm quality and quantity may have a dramatic impact on female fecundity and fertilization success. Despite this, the relationships between these parameters are rarely investigated. Moreover, studies exploring age-related variation in male mating success generally fail to consider the entire lifespan of an individual male; instead they restrict analyses to a small number of defined age classes. As a consequence they are unable to assess the impact of early and late life history components on male reproductive success. In this study, we explore these questions using the hide beetle, Dermestes maculatus , a model species for investigating age-related male mating success. We first employed a longitudinal design to explore whether patterns of sperm transfer and subsequent reproductive success varied over the reproductive lives of a cohort of males. Secondly, we investigated age-related variation in sperm viability, a surrogate measure of sperm quality. Finally, we combined these data and assessed whether the observed patterns of sperm transfer were correlated with fertilization success. We found that the quantity of sperm varied with male age: the amount of sperm transferred to a female increased with male age until 9 wk and then started to decline. Similarly, female fecundity and fertilization success were related to male age: females mating with males when they were relatively young (1 wk) or relatively old (13 wk) suffered reductions in fecundity and fertilization. Our data suggest that fertilization success is driven at least in part by the quantity of sperm transferred during mating.     

Female reproductive behaviour,ovarian hormones and vaginal cytology of the induced ovulator, <Emphasis Type="Italic">Ctenomys talarum</Emphasis>

In the present study, we evaluated whether reproductive condition affects female reproductive behaviour in the induced ovulator Ctenomys talarum. We also explored the effect of the interaction with a male on the reproductive condition of females. To evaluate this, we arranged mating trials and evaluated female reproductive behaviour. Reproductive status of females was evaluated using a combined approach of vaginal smears, urinary progesterone and oestradiol, and ovarian histology. Behaviours denoting attraction (‘male sniff’ and ‘mount attempts’) and mutual courtship behaviours (‘spin’ and copula) were correlated with vaginal cytology before and oestradiol and progesterone levels in urine 12 h after male–female encounter. After 24 h of the interaction, oestradiol levels and vaginal epithelization increased while progesterone levels decreased in soliciting females. C. talarum females’ reproductive behaviour was related to its physiological reproductive state and vaginal cytology. The kind of male interaction, whether couples copulated or remained indifferent affected the later status of females. Females are induced ovulators by mating but male presence and interaction also affected other components of their reproductive physiology such as ovarian hormones and vaginal cytology.     

Lack of sex‐specific movement patterns in an alien species at its invasion front – consequences for invasion speed

Efficient targeting of actions to reduce the spread of invasive alien species relies on understanding the spatial, temporal, and individual variation of movement, in particular related to dispersal. Such patterns may differ between individuals at the invasion front compared to individuals in established and dense populations due to differences in environmental and ecological conditions such as abundance of conspecifics or sex‐specific dispersal affecting the encounter rate of potential mates. We assessed seasonal and diurnal variation in movement pattern (step length and turning angle) of adult male and female raccoon dog at their invasion front in northern Sweden using data from Global Positioning System (GPS)‐marked adult individuals and assessed whether male and female raccoon dog differed in their movement behavior. There were few consistent sex differences in movement. The rate of dispersal was rather similar over the months, suggesting that both male and female raccoon dog disperse during most of the year, but with higher speed during spring and summer. There were diurnal movement patterns in both sexes with more directional and faster movement during the dark hours. However, the short summer nights may limit such movement patterns, and long‐distance displacement was best explained by fine‐scale movement patterns from 18:00 to 05:00, rather than by movement patterns only from twilight and night. Simulation of dispersing raccoon dogs suggested a higher frequency of male–female encounters that were further away from the source population for the empirical data compared to a scenario with sex differences in movement pattern. The lack of sex differences in movement pattern at the invasion front results in an increased likelihood for reproductive events far from the source population. Animals outside the source population should be considered potential reproducing individuals, and a high effort to capture such individuals is needed throughout the year to prevent further spread.