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Pii: s0264-410x(00)00183-3

Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis Juan M. Torresa,*, Miguel A. RamõÂreza, MoÂnica Moralesa, Juan BaÂrcenaa, BeleÂn VaÂzqueza, Enric EspunÄab, Albert PageÁs-ManteÂb, Jose M. SaÂnchez-VizcaõÂnoa aCentro de InvestigacioÂn en Sanidad Animal (CISA-INIA), Valdeolmos, 28130 Madrid, Spain Received 14 February 2000; received in revised form 9 May 2000; accepted 12 May 2000 We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [BaÂrcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114±23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its ®eld use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable e€ects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Safety; Myxoma-RHDV; Transmissible vaccine since the deliberate release of MV in France (1952) as a biological control agent of wild rabbit populations.
Myxomatosis and rabbit haemorrhagic disease Immunization of domestic rabbits against myxomato- (RHD) are considered the major viral diseases a€ect- sis is currently achieved using heterologous vaccines ing the European rabbit (Oryctolagus cuniculus ). Myx- based on Shope ®broma virus, a less virulent Lepori- oma virus (MV), the causative agent of myxomatosis, poxvirus, or homologous vaccines based on cell cul- belongs to the Leporipoxvirus genus of the Poxviridae ture-attenuated strains of MV [4,5].
family [1]. In its natural host, Sylvilagus rabbits in the RHD was ®rst reported in the People's Republic Americas, MV induces a mild benign infection. In of China [6]. The disease spread throughout Europe European rabbits however, MV causes the systemic between 1987 and 1989 [7] and is endemic since and lethal infection known as myxomatosis [2,3]. The then. Infected rabbits usually die within 48±72 h of disease is endemic in the entire rabbit range in Europe necrotising hepatitis. RHD is responsible for high economic losses in rabbitries as well as high mor- tality rates in wild rabbit populations [8±12]. The * Corresponding author. Tel.: +34-91-620-23-00; fax: +34-91-620- etiological agent, rabbit haemorrhagic disease virus E-mail address: (J.M. Torres).
(RHDV), is a member of the Caliciviridae family 0264-410X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
J.M. Torres et al. / Vaccine 19 (2001) 174±182 [13]. The RHDV virions are non-enveloped and ico- recombinant 6918VP60-T2 virus concerning the sahedral, with capsids composed of a major protein component of 60 kDa (VP60). Commercial vaccines against RHD are prepared from the livers of exper- imentally infected rabbits [14], since in vitro systems are not available for ecient virus propagation. In the last years, the VP60 gene has been successfully expressed in several heterologous systems [15±23] and has been shown to induce full protection of Recombinant virus 6918VP60-T2 was propagated in rabbits against a lethal challenge with RHDV.
RK-13 (rabbit kidney) cell line grown in Dulbecco's While the currently available vaccines against minimum essential medium (DMEM) supplemented myxomatosis and RHD have proven e€ective in the with 5% foetal bovine serum (FBS), 2 mM L-gluta- control of these diseases in domestic rabbits, they mine, 100 U/ml penicillin, and 100 mg/ml streptomycin.
are not suited to immunize wild rabbit populations, SIRC (rabbit cornea) cells were used for viral titre de- as vaccines need to be delivered individually by termination on plaque assay. Both rabbit cell lines conventional veterinary practices, which is not a were obtained from the American Type Culture Col- feasible approach to vaccinate free ranging animals.
As a novel approach for wildlife vaccination, we have explored the possibility of developing ``trans- missible vaccines'' by the use of viral vectors capable of spreading within an animal population.
Common rabbits (brown coloured) free from anti- In order to protect wild rabbits against both myxo- MV and anti-RHDV antibodies, were provided by a matosis and RHD, we constructed a recombinant commercial breeder. These rabbits are routinely used virus based on the naturally attenuated MV ®eld for restocking in the ®eld and from now on will be strain 6918 [24], that expressed the RHDV VP60 protein [25]. A linear epitope tag from the nucleo- protein of porcine transmissible gastroenteritis coro- 2.3. Administration of an overdose of 6918VP60-T2 recombinant VP60 protein to allow monitoring the spread of the recombinant virus in the environment.
Groups of eight wild rabbits (2 month old, weighing Following inoculation of rabbits, the recombinant around 0.8 kg) free from MV and RHDV antibodies, virus (6918VP60-T2) induced speci®c antibody re- were inoculated at the back by intradermic (i.d.) or sponses against MV, RHDV as well as for the subcutaneous (s.c.) route with di€erent doses of the TGEV tag. Administration of 6918VP60-T2 by the vaccine (104, 105, 106 pfu of 6918VP60-T2 recombinant subcutaneous, intradermal or oral routes protected virus). Rabbits were observed daily for 21 days and rabbits against lethal RHDV and MV challenges.
clinical symptoms were recorded. Weight and tempera- Furthermore, the recombinant 6918VP60-T2 virus ture determinations were made on each animal until showed a limited horizontal transmission capacity, the 21st day. Serum samples extracted from the mar- either by direct contact or in a ¯ea-mediated pro- ginal ear vein of the rabbits on days 0 and 21 after im- cess, promoting immunization of contact uninocu- munization were used to evaluate the serological responses against MV and RHDV, by using an The promising results obtained so far under lab- enzyme-linked immunosorbent assay (ELISA), as pre- viously described [25]. Antibody titres were de®ned as 6918VP60-T2 could be used in large-scale immuniz- the reciprocal of the highest dilution giving an A405 ation schemes for the control of myxomatosis and value two-fold over the background level (negative RHD in wild rabbit populations. However, before considering its environmental release, vaccine safety considerations should be extensively evaluated. Poten- 2.4. Administration of 6918VP60-T2 virus to tial risks with regard to vaccine dose (i.e., accidental administration of an overdose), age, physiological condition (i.e., pregnant does) and immune status of Groups of eight wild rabbits (2 month old, weighing exposed individuals, should be taken into account.
around 0.8 kg) were immunosuppressed by treatment Biological stability is another important aspect to with prednisolone (2 mg per animal per day) for 3 evaluate in a recombinant virus intended for environ- days before vaccination and 2 days after vaccination.
mental release. In the present study, we report the Prednisolone treated rabbits were inoculated by i.d. or safety evaluation under laboratory conditions of s.c. route at the back with 104 pfu of 6918VP60-T2 J.M. Torres et al. / Vaccine 19 (2001) 174±182 virus. Control rabbits were vaccinated but not treated with prednisolone. Rabbits were observed daily for a period of 21 days and clinical symptoms were Data were analysed using a Student's t-test for non- recorded. Weight and temperature determinations were paired variants. Signi®cance was considered when p ` made on each animal until the 21st day. Serum samples extracted 0 and 21 days after immunization were used to evaluate the serological responses against MV and RHDV by ELISA. Antibody titres were 3.1. E€ects induced by the administration of an overdose 2.5. Administration of 6918VP60-T2 virus to pregnant Previous work showed 104 pfu was an ecient vac- cine dose to ensure horizontal transmissible protection Groups of six pregnant does were inoculated at against myxomatosis and RHD, either by direct con- di€erent times of gestation (days 7, 14, 21 and 28) by tact or in a ¯ea-mediated process [25]. To evaluate the s.c. route at the back with 104 pfu of 6918VP60-T2 e€ects of administering an overdose of the vaccine, virus. Control does were inoculated at the same days wild rabbits were inoculated by i.d. or s.c. route with of gestation with 0.5 ml of phosphate-bu€ered saline di€erent doses of 6918VP60-T2 virus (104, 105 and 106 (PBS). Animals were observed daily and general clini- cal symptoms were recorded. No body weight and In order to obtain a semi-quantitative measure to temperature determinations were performed in order allow graphic representation and objective comparison, to minimise the handling-induced stress in does, which the classical myxomatosis symptoms were classi®ed in are specially sensible during gestation. The following a ranking of 1 to 6 points (see Table 1), and the results reproductive parameters were recorded both at ®rst registered during the observation period were rep- and second parturition: number of animals born alive resented (Fig. 1). Rabbits inoculated by i.d. route dis- per litter; number of animals born dead per litter; played similar clinical signs at all vaccine doses tested.
number of living animals per litter 8 days postparturi- These consisted of a localised primary nodule at the tion (dpp), and weight of each litter at 8 dpp.
inoculation site and, in some rabbits, scanty secondary skin lesions in the form of small discrete nodules, usually less than 0.5 cm in diameter, in face, ears or 2.6. Analysis of 6918VP60-T2 virus biologic stability eyelids. Lesions appeared 5±7 days postinoculation (dpi) and completely resolved in all rabbits normally Two rabbits (2 month old, weighing around 0.8 kg) by 15 dpi. None of the infected rabbits exhibited clas- were inoculated by i.d. route at the back with 104 pfu sical severe myxomatosis symptoms like closure of the of 6918VP60-T2 virus. Seven to 9 days postvaccination eyes, generalised oedema, or respiratory syndrome the inoculation site nodule was extracted, homogen- (Fig. 1). Rabbits inoculated by s.c. route showed simi- ated in PBS, and reinoculated into two new rabbits.
lar clinical symptoms but these were consistently This procedure was repeated up to 10 passages. The virus obtained from the last passage was titrated and the e€ects of inoculating 104 pfu by s.c. in a group of eight rabbits were evaluated as described above and compared with those of the original recombinant virus.
Serum samples extracted 0 and 21 days after immuniz- ation were used to evaluate the serological responses against MV and RHDV by ELISA. Antibody titres were de®ned as described above. In order to evaluate the genetic stability of 6918VP60-T2 virus after 10 pas- sages in rabbits, DNA extracted from the nodules at the inoculation site was analysed by PCR. The oligo- nucleotides used were MV1 and MV2, which are de- rived from the MV genomic sequence ¯anking the foreign gene insertion site [25]. The ampli®cation of a Fig. 1. E€ects of administering di€erent doses of 6918VP60-T2 virus.
3.3-kb PCR product, instead of the 1.0-kb product Groups of eight wild rabbits were inoculated by i.d. route with 104 (*), 105 (Q), or 106 (R) pfu. Rabbits were observed daily for a obtained from wild-type MV, was indicative of the period of 18 days and the clinical signs due to virus infection of each presence of the inserted VP60 gene construct.
animal were ranked from 0 to 6 according to Table 1.
J.M. Torres et al. / Vaccine 19 (2001) 174±182 Value assignment of the di€erent clinical signs developed by rabbits in the course of a myxomatosis infection A localised primary nodule at the inoculation site Secondary skin lesions in the form of small discrete nodules near the inoculation site, in face, or ears Small nodules in genitals, limbs, and other parts of the body Severe myxomatosis symptoms like closure of the eyes, generalised oedema, or respiratory syndrome milder: there were less secondary nodules, which were with 104 pfu of 6918VP60-T2 virus, and clinical signs slightly smaller and resolved earlier (results not due to virus infection were compared with those shown). No febrile response or loss of body weight induced in control rabbits, which were vaccinated but was detected. Table 2 shows temperature increases not treated with prednisolone (Fig 2, Table 3). Results registered from 0 to 2 dpi and from 0 to 4 dpi, as well indicated that administration of 6918VP60-T2 virus to as the weight increase from day 0 to day 21. No sig- immunocompromised animals was safe (either by i.d.
ni®cant di€erences in the increases of body tempera- or s.c routes), as prednisolone treated rabbits exhibited ture or body weight were observed in recombinant only mild clinical symptoms and were all completely virus-infected rabbits as compared with control rab- recovered by 18 dpi. Fig. 2 shows a graphic represen- bits, regardless of virus dose or inoculation route.
tation of the symptomatology observed in rabbits To evaluate the immune responses elicited by the inoculated by i.d. route, according to the ranking of inoculated rabbits, sera samples obtained 21 dpi were myxomatosis clinical signs established in Table 1.
monitored by ELISA for the presence of anti-MV and After i.d. inoculation, immunosuppressed rabbits anti-RHDV antibodies. The inoculated rabbits devel- exhibited similar local lesions to those observed in con- oped high anti-MV and anti-RHDV antibody titres, trol non-immunosuppressed rabbits. Lesions appeared which increased with the vaccine dose (Table 2). There at the same time (5±7 dpi) in both cases but showed a was no gross di€erence in the antibody titres induced subtle tendency to resolve later in immunosuppressed by vaccine administration by i.d. or s.c. inoculation rabbits (15±18 dpi vs. 15 dpi). Results obtained with rabbits inoculated by the s.c route were essentially the same (data not shown). No signi®cant di€erences in 3.2. E€ects induced by the administration of 6918VP60- body temperature increase or body weight increase were observed when immunosuppressed rabbits were compared with control rabbits (Table 3). The humoral To evaluate the e€ects of recombinant virus infec- immune responses elicited 21 dpi in both prednisolone tion on immunocompromised animals, rabbits were treated and control rabbits were similar. All vaccinated immunosuppressed by treatment with prednisolone.
rabbits developed high anti-MV and anti-RHDV anti- Treated rabbits were inoculated (by s.c or i.d. route) E€ects of one overdose of the vaccine (6918VP60-T2) Vaccination route Mean body temperature increase Mean body weight increase Mean antibody titres Vaccinated with 10 doses (105 pfu) s.c.
Vaccinated with 10 doses (105 pfu) i.d.
Vaccinated with 100 doses (106 pfu) s.c.
Vaccinated with 100 doses (106 pfu) i.d.
J.M. Torres et al. / Vaccine 19 (2001) 174±182 Fig. 2. E€ects of administering 6918VP60-T2 virus to immunosup- Fig. 3. E€ects of administration of 6918VP60-T2 virus after 10 pas- pressed rabbits. Groups of eight rabbits treated (R) or untreated (Q) sages in vivo. Groups of eight rabbits were inoculated by s.c. route with prednisolone were inoculated by i.d. route with 104 pfu of with Passage 0 (Q) or Passage 10 (R) 6918VP60-T2 virus. Rabbits 6918VP60-T2 virus. Rabbits were observed daily for a period of 18 were observed daily for a period of 18 days and the clinical signs days and the clinical signs due to virus infection of each animal were due to virus infection of each animal were ranked from 0 to 6 ranked from 0 to 6 according to Table 1.
3.3. E€ects induced by the administration of 6918VP60- virus-infected does showed any symptomatology as- To evaluate the e€ects of recombinant virus infec- 3.4. Analysis of the biological stability of 6918VP60-T2 tion on reproduction, pregnant does were inoculated at di€erent times of gestation (days 7, 14, 21 and 28) by s.c. route. The daily observation of the animals The biological stability of the recombinant virus, showed a total absence of general clinical symptoms in and therefore its potential to evolve to a virulent state all inoculated animals. Reproductive parameters such were evaluated by comparing the e€ects of rabbit as number of animals born alive per litter, number of infection with ``Passage 0'' virus (the same virus stock animals born dead per litter, number of living animals used in all the experiments reported in this paper), per litter 8 dpp, and average weight of each litter at 8 with the e€ects of rabbit infection with the virus dpp, for both ®rst and second parturition, have been obtained after 10 serial passages in rabbits (Passage 10 summarised in Table 4. The overall results showed virus). Fig. 3 shows a graphic representation of the that recombinant virus infection did not induce any symptomatology observed in rabbits infected with alteration during reproduction. Pregnant does infected either Passage 0 or Passage 10 virus, according to the at di€erent days of gestation showed reproductive ranking of myxomatosis clinical signs established in values being in the expected range for rabbits, and no Table 1. Rabbits infected with Passage 10 virus exhib- di€erences were observed when recombinant virus- ited the same mild clinical signs as those infected with infected does were compared with control does inocu- Passage 0 virus. Symptoms appeared 5±7 dpi and com- lated with PBS at the same day of gestation. The pletely resolved by 15 dpi in both cases. None of the absence of alterations in reproductive parameters was infected rabbits exhibited classical severe myxomatosis maintained in the following parturition (Table 4). Fur- symptoms. Table 5 shows temperature increases from thermore, none of the rabbits born from 6918VP60-T2 0 to 2 dpi and from 0 to 4 dpi, as well as weight E€ects induced by 6918VP60-T2 virus infection in immunosuppressed rabbits Vaccination route Mean body temperature increase Mean body weight increase Mean antibody titre J.M. Torres et al. / Vaccine 19 (2001) 174±182 increases from day 0 to 21. No signi®cant di€erences oral vaccination is being used to control enzootic syl- in body temperature increase or body weight increase vatic rabies in Europe and North America by means were observed when rabbits infected with Passage 10 of a recombinant vaccinia-rabies vaccine delivered by virus were compared with rabbits infected with Passage baiting [26]. An alternative strategy is the use of 0 virus or control uninfected rabbits. The humoral re- ``transmissible vaccines'', i.e., viral vectors capable of sponses elicited by rabbits infected with Passage 0 or spreading within an animal population. Hopefully, the Passage 10 virus were similar. All infected rabbits administration of a recombinant vaccine of this developed high anti-MV and anti-RHDV antibody characteristics to a small number of captured individ- uals, would eventually lead to the immunization of a The genomic stability of 6918VP60-T2 virus was fraction of animals within a given population, which is analysed by PCR using oligonucleotide primers exter- sucient to reduce the spread of the target disease.
nal to the insertion site of the VP60 gene. After 10 This approach might be useful, especially when the dis- serial passages in rabbits, a product of 3.3 kb (the tribution, size, and turnover rate of a population pre- expected size for the recombinant virus) was ampli®ed cludes capture or baiting techniques as the only means by PCR with no detection of the corresponding wild- for antigen delivery. The European rabbit is an type MV 1.0 kb product (not shown), indicating that example of such a population. With this in mind, we the VP60 gene was stably integrated in the MV gen- have developed a transmissible vaccine against both myxomatosis and RHD based on a recombinant MV- VP60 virus capable of spreading through rabbit popu- lations [25]. The results obtained under laboratory conditions suggest the recombinant virus might be e€ective for wild rabbit immunization. However, since A number of vaccines are available to protect rab- the proposed use of 6918VP60-T2 involves the en- bits against myxomatosis and RHD [4,5,14] which are vironmental release of a recombinant virus, consider- useful for immunizing domestic rabbits. However, con- ations regarding safety issues are as important as the trol of both diseases among wild rabbit populations potential ecacy of the candidate vaccine. It is for this remains an unsolved problem of great concern. In this reason that safety concerns have been at the core of regard it should be noted that the European rabbit the rational design of the proposed immunization plays a key ecological role in Mediterranean ecosys- tems. In addition, rabbits are among the most import- The biological characteristics of MV make it a ant small game species in several European countries.
good candidate as a vaccine vector in terms of Immunization of wildlife is dicult to achieve safety considerations. MV exhibits a very restricted because direct delivery of vaccines to free ranging ani- host range, infecting exclusively rabbits (both Sylvi- mals is not possible. The oral route is considered a lagus and Oryctolagus spp.). The virus has been feasible way of vaccine administration. For example, widely distributed throughout Europe, Australia and E€ects induced by 6918VP60-T2 virus infection in pregnant does J.M. Torres et al. / Vaccine 19 (2001) 174±182 the Americas for nearly 50 years with no evidence of infection of other species. Thus, the host restricted nature of MV minimises the risk of recombinant vaccine spreading to non-target species in nature. On the other hand, given the current widespread geographic distribution of MV, which is similar to the distribution of RHDV, the ®eld use of a recombinant MV-VP60 vaccine would normally not involve the introduction of a virus species that does not already exist in a particular area.
Safety aspects were also considered in the choice of the parental MV strain. It was decided not to use one of the available vaccinal strains, obtained by cell culture-attenuation of virulent MV strains [5], as this would involve the release of a new strain to the environment, which might undergo reversion to virulence in nature. Instead, we decided to use an attenuated MV ®eld strain which was already circulating among wild rabbit populations.
Strain 6918 was selected from a ®eld survey of MV strains circulating in Spain, which were analysed for virulence and transmissibility [24]. This strain exhib- ited adequate biological characteristics for the devel- opment of a recombinant transmissible vaccine, as it caused a non-pathogenic infection comparable to that of cell culture-attenuated vaccinal strains, yet retaining the capacity of horizontal spreading [24].
Since preservation of the valuable biological prop- erties of 6918 strain was of major importance in the development of the recombinant virus, the foreign gene was inserted in the intergenic site between ORFs MJ2 and MJ2a, as recombinant MVs with insertions at this site have been shown to retain overall parental biological characteristics [27]. Moreover, the VP60 expression cassette was inserted into the MV genome using the TDS two- step selection system [28]. This procedure enables the construction of recombinant poxviruses without any marker genes inserted in the ®nal recombinant viral genome. Thus, the recombinant 6918VP60-T2 does not harbour selectable markers such as anti- biotic resistance genes, the widespread of which is currently regarded as a major health and environ- mental threat. Considering the potential risks associ- ated with the DNA sequence inserted, it should be noted that the VP60 gene has been cloned in a wide range of heterologous systems[15±23] and no indication of toxicity or side e€ects associated to the expression of VP60 have been reported.
Previous results indicated that administration of either 6918 MV or recombinant 6918VP60-T2 virus to healthy rabbits under laboratory conditions by stan- dardised procedures is safe, as all rabbits exhibited only mild clinical symptoms and rapidly recovered [24,25]. In this report we have extended the safety assessment of the vaccine by analysing the potential J.M. Torres et al. / Vaccine 19 (2001) 174±182 risks of vaccine administration under a varied range of On the basis of the results previously reported situations that might occur if the recombinant virus is [24,25] and those presented in this paper, along with used for large-scale ®eld immunization of rabbits.
experimental data addressing further safety and e- Concerning vaccine dosage and the possibility of cacy issues (to be published elsewhere), the recombi- accidental administration of an overdose, the results nant 6918VP60-T2 has been subjected to the demonstrated vaccine safety even when a 100-fold mandatory risk assessment process relative to the overdose (106 PFU) was inoculated (Fig. 1, Table 2).
release of genetically-modi®ed organisms. A limited Assessment of vaccine e€ects in immunosuppressed ®eld trial authorised by the Spanish competent auth- rabbits was considered relevant, given the incidence in orities is in course. This trial will assess the ecacy nature of immunocompromised individuals due to and safety of the vaccine under controlled ®eld con- infections, environmental or genetic causes. For this ditions, in the perspective of its use in a large-scale reason we assayed the e€ect of vaccine administration program for the control of myxomatosis and RHD in rabbits treated with prednisolone, a potent immuno- suppressor. This treatment induces depletion of circu- lating eosinophils and mononuclear cells, causing a strong decrease of the T-cell response with only a slight e€ect on B-cell function [29]. It is a commonly used procedure for the safety evaluation of veterinary This work was supported by an agreement between vaccines [30±32]. Results showed that prednisolone the ``FundacioÂn para el Estudio y Defensa de la Nat- treated rabbits exhibited similar symptoms to those uraleza y la Caza'' (FEDENCA) and the ``Instituto observed in control rabbits (Fig. 2, Table 3). The only Nacional de InvestigacioÂn y TecnologõÂa Agraria y Ali- remarkable observation was that immunosuppressed rabbits showed a subtle tendency to delay the resol- ution of local lesions: 16±18 dpi vs. 15 dpi (Fig. 2).
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Battery care & tips card

Battery Care & Tips Radio Products and Services Division These battery tips will help you obtain optimized performance and a longer life cycle from your Motorola rechargeable battery. 1. Charge your new battery overnight before using it. This is referred to as INITIALIZING and will enable you to obtain maximum battery capacity. a. Nickel Cadmium or Nickel Metal Hydrid

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Clinical features and outcome in dogs and cats with obsessive-compulsive disorder: 126 cases (1989–2000) Karen L. Overall, VMD, PhD, DACVB, and Arthur E. Dunham, PhDning, fly biting, self mutilation, hair or air biting, pica,pacing or spinning, staring and vocalizing, some aggres- Objective —To determine clinical features and out- sions, self-directed vocalizing, and fabric suckin

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