Vesicular exanthema of swine and San Miguel sea lion virus: experimental and field studies in otarid seals, feeding trials in swine

The naturally occurring disease caused by San Miguel sea lion virus in fur seals was characterized by small fluid-filled vesicles 1 to 25 mm in diameter on the nonhaired portions of the flippers. Early epithelial lesions contained multifocal sites of cell lysis. The resultant microvesicles enlarged and coalesced, forming grossly visible macrovesicles. Mature vesicles progressed to involve all layers of the epithelium but did not involve the underlying dermis. Intradermal inoculation of vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2 into otarid (fur) seal pups caused plaque-like lesions around inoculated coronary bands. These swellings regressed without rupture by 96 hours postinoculation. One seal inoculated with San Miguel sea lion virus had a linear lingual erosion at ten days postinoculation. Virus was isolated from this site and from two uninoculated sites, the tonsil and testicle. Contact controls showed no evidence of infection. Virus was isolated in low titers from some sites of inoculation and draining lymph nodes from seals infected with vesicular exanthema of swine virus. Virus was recovered more easily, in higher titers, and from more tissues, from seals infected with San Miguel sea lion virus. Inoculated seals tested after four to ten days seroconverted. Feeding swine seal tissues from the inoculation experiments resulted in seroconversion in swine which were fed tissues from seals infected with vesicular exanthema of swine virus but not in those which were fed tissues from seals infected with San Miguel sea lion virus.     

Current prevalence of adult Uncinaria spp. in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, California, with notes on the biology of these hookworms

A prevalence survey for hookworms (Uncinaria spp.) was done in northern fur seal (Callorhinus ursinus) and California sea lion (Zalophus californianus) pups on San Miguel Island, CA, in 2000. Intestines of dead pups were examined for adult hookworms in July. These parasites were found in 95% of 20 fur seal pups and 100% of 31 sea lion pups. The number of hookworms varied from 4 to 2142 (mean = 760) in fur seal pups and from 20 to 2634 (mean = 612) in sea lion pups. A direct relationship was evident between body condition and number of hookworms in the pups; that is, pups in poor condition had fewer hookworms than those in good condition. There was a decline in the number of hookworms in sea lion pups in 2000 compared to collections in 1996. Eggs of Uncinaria spp. were found in rectal feces (collected in late September and early October) of none of 35 (0%) live fur seal pups and 41 of 48 (85%) live sea lion pups. Packed cell volume values, determined for most of the same live pups, were essentially normal for C. ursinus but were much lower than normal for most Z. californianus. Hookworm larvae were not found in blubber of fur seal and sea lion pups or in rookery sand in July. Rookery sand, positive for live hookworm larvae when put in a refrigerator, was negative at removal 2.5 years later. The average number of eggs in utero of female hookworms was 285 for three specimens from a fur seal pup and 281 from three specimens from a sea lion pup. One hookworm larva was recovered from milk stripped from the teats of a stranded Z. californianus female at The Marine Mammal Center, Sausalito, CA.     

San Miguel sea lion virus fed to mink and pigs.

Mink became infected with San Miguel sea lion virus when fed ground meat from seal carcasses showing vesicular-like lesions in the skin. The mink also contracted the infection when they were fed San Miguel sea lion virus infected pig meat or cell culture propagated virus. San Miguel sea lion virus infection in mink was inapparent but the virus was isolated from blood and rectal swabs. Pigs treated similarly with the same virus preparations given to mink developed a severe vesicular disease syndrome similar to that produced by vesicular exanthema of swine. In a separate trial, pigs fed a large sample of commercial ground seal meat did not develop disease signs or antibodies. Further work is needed to assess the hazard of introducing San Miguel sea lion virus into swine on the same premises when potentially San Miguel sea lion virus infective seal meat is fed to mink.     

Isolation of San Miguel Sea Lion Virus from Samples of an animal food product produced from northern fur seal (Callorhinus ursinus) carcasses

A virus was isolated from California sea lions (Zalophus californianus californianus) and northern fur seals (Callorhinus ursinus) in 1972. It was later named San Miguel sea lion virus (SMSV). State and federal livestock disease control agencies became concerned, because SMSV was found to be indistinguishable from vesicular exanthema of swine virus and to cause (in laboratory trials) clinical signs in swine similar to those produced by vesicular exanthema of swine virus. Ground carcasses of northern fur seals, salvaged after harvesting pelts, are fed to mink on ranches in the United States. Domestic swine are kept on some of these same ranches. Samples withheld from lots of this seal carcass mink food were found to contain SMSV (serotype 5) in titers of 10(6.1) and 10(6.8) tissue culture infective doses.     

Host range comparisons of five serotypes of caliciviruses

Two new serotypes of San Miguel sea lion virus (SMSV), designated SMSV-4 and SMSV-5, were studied in vivo and in vitro. The host cell spectrums were compared with SMSV-1, SMSV-2, and vesicular exanthema of swine virus type A-48. Based on the result of these broad host spectrums, a numerical scoring system was devised for ranking each virus on the basis of its potential for infecting terrestrial mammals, including the important domestic species.     

The pathogenesis of vesicular exanthema of swine virus and San Miguel sea lion virus in swine

Vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2, when inoculated intradermally into swine, resulted in fluid-filled vesicles at the sites of inoculation in the snout, coronary band, and tongue. Pigs that developed vesicles also had fevers. Secondary vesicle formation varied, depending on virus serotype. Viremia was found in one pig infected with San Miguel sea lion virus five days after infection. Virus was recovered from nasal-oral passages for up to five days after infection in both groups of pigs and from the gastrointestinal and urinary tracts of pigs infected with San Miguel sea lion virus. Neutralizing antibodies began to increase three days after inoculation and reached peak titers in seven to ten days. In the absence of secondary bacterial infection, healing was well advanced by ten days after inoculation. Lesions usually were limited to nonhaired portions of the integument and tongue. Individual epithelial cells became infected when a break in the skin allowed virus access to susceptible epithelial cells from either exogenous or endogenous sources. Individual infected cells ruptured and adjacent cells were infected, resulting in the formation of multiple microvesicles. Centrifugal coalescence of microvesicles led to formation of grossly visible macrovesicles. Lesions rarely developed from viral contamination of intact hair follicles. A mild virus-induced encephalitis was seen in pigs infected with vesicular exanthema of swine virus, and virus was recovered from brain tissue of pigs infected with San Miguel sea lion virus.     

Urate nephrolithiasis in a northern elephant seal (Mirounga angustirostris) and a California sea lion (Zalophus californianus).

Nephrolithiasis has rarely been reported in marine mammals. During 2004 and 2005, two cases of nephrolithiasis were diagnosed during routine necropsy examination, one in a northern elephant seal (Mirounga angustirostris) and one in a California sea lion (Zalophus californianus). Nephroliths were found throughout both kidneys during necropsy examination, varying in size from 1-10 mm in diameter in the northern elephant seal and from 1-15 mm in diameter in the California sea lion. Necropsy and histopathology revealed nephroliths in association with renal pelvic dilation and pyelonephritis in both animals. In addition, hydronephrosis was noted in the sea lion. Nephroliths were composed of uric acid and ammonium urate in the northern elephant seal and of ammonium urate in the California sea lion. The underlying disease leading to nephrolith formation was not determined; however, it is hypothesized that unknown metabolic derangements due to morphologic or physiologic differences may have played a role. This is the first report of urate nephrolithiasis in the California sea lion and northern elephant seal.     

Experimental infection of pups with Ancylostoma caninum-infected mouse tissues

Migration and distribution of Ancylostoma caninum larvae in the tissues of mice orally infected with 1000 larvae, and establishment of patent infection from the mice to the definitive host, were studied. Larval yield from different organs of mice, after digestion with artificial gastric juice, indicated that the highest recovery was at 4 h post-infection (62.8%), and thereafter a slight decline occurred up until 30 days post-infection (51.5%). Migration of larvae to the lungs occurred within 4 h, to the liver within 12 h and into the heart within 24 h. No larvae were recovered from spleen and kidney tissues. From the 9th day onwards larvae were also recovered from the brain. Migration in the muscles of head and neck occurred within 4 h, in the thoracic and abdominal muscles at 24 h and in lumbosacral and leg muscles at 48 h. The establishment of patent infections in the definitive host was studied by feeding the orally- and percutaneously-infected mice to hookworm-free pups at 10 and 30 days post-infection. The mean necropsy worm burden in the pups fed with the orally-infected mice was comparatively higher than in the pups fed cutaneously-infected mice.     

The transmission of phocine herpesvirus-1 in rehabilitating and free-ranging Pacific harbor seals (Phoca vitulina) in California

Phocine herpesvirus-1 (PhHV-1) causes regular outbreaks of disease in neonatal harbor seals (Phoca vitulina) at rehabilitation centers in Europe and in the U.S. To investigate transmission of this virus samples were collected from harbor seal pups during exposure studies at a Californian rehabilitation center from 1999 to 2002 and from free-ranging harbor seals off central California during the same period. The exposure studies provided evidence that PhHV-1 can be transmitted horizontally between animals most likely through direct contact with oro-nasal secretions. However vertical transmission may also occur, as adult female harbor seals were found to be shedding the virus in vaginal and nasal secretions, and premature newborn pups had evidence of early infection. Results also indicated that PhHV-1 infections were common in both free-ranging (40%, 49/121) and rehabilitating (54%, 46/85) young harbor seals, during the spring and early summer. This timing, which correlated with pupping and weaning, suggested that the majority of animals were infected and infective with PhHV-1 between pupping and breeding.     

Transmission of Loma salmonae (Microsporea) to chinook salmon in sea water.

Transmission studies were conducted to determine if Loma salmonae was transmissible in sea water. Transmission of L. salmonae to chinook salmon (Oncorhynchus tshawytscha) held in sea water was achieved by exposing fish to macerated, infected gill tissue. Fish were exposed in seawater in a flow-through aquarium, and the infection was detected as soon as 5 wk after exposure. Heavily infected fish exhibited numerous xenomas in the branchial arteries, central venous sinusoids, and within the blood channels of the lamellae. The pathological changes were similar to those seen in pen-reared salmon with L. salmonae infections. The infection was not observed in Atlantic salmon (Salmo salar), Pacific herring (Clupea pallasi, family Clupeidae), or shiner perch (Cymatogaster aggregata, family Embiotocidae), experimentally exposed using identical methods. This study suggests that L. salmonae is transmissible to chinook salmon in seawater netpens. Fish farmers and fish health specialists should consider this possibility when developing and implementing strategies to control the infection.     

Experimental and naturally occurring transplacental transmission of canine distemper virus

In the present study, 2 different effects of experimentally induced infection with virulent canine distemper virus (CDV) on pregnant CDV-susceptible dogs were studied. In 1 bitch, abortion occurred 7 days after viral inoculation and there was no evidence of fetal infection. Another bitch had subclinical infection and delivered 3 CDV-infected pups. Sequential clinical, immunologic, and virologic studies of a litter of gnotobiotic pups (3rd bitch) that were congenitally infected with CDV demonstrated the heightened susceptibility to CDV in the neonatal period. The data presented add canine distemper to the list of transplacental infectious diseases in the canine species.     

Serological survey of pre-weaned New Zealand fur seals (Arctocephalus forsteri) for brucellosis and leptospirosis

AIM: To conduct a longitudinal serological survey for evidence of Brucella spp and Leptospira spp infection of pre-weaned New Zealand fur seals in a colony on the Otago Peninsula. METHODS: Seal pups were repeatedly captured on a monthly basis from February through to July 2001. Pups were tagged at first capture and a blood sample was taken at each capture event. A total of 163 sera were collected from 118 seal pups. Where sufficient volume was collected, the sera were tested for leptospirosis using the microscopic agglutination test (MAT), and for brucellosis using the competitive enzyme-linked immunosorbent assay (ELISA) for Brucella abortus. RESULTS: None of 128 sera from 101 seals tested positive to the ELISA for B. abortus. All tests for Leptospira interrogans serovars Grippotyphosa, Copenhageni, Bratislava and Leptospira borgpetersenii serovar Ballum were negative at a cut-off of <1/100 dilution. Positive or suspicious titres were found to L. interrogans serovars Canicola and Pomona and L. borgpetersenii serovar Hardjo. The highest titres (12,800) were found to serovar Pomona. The titre to serovar Pomona in one seal rose from <1/50 in March to 12,800 in April and was <1/50 when re-sampled in July. The titre to serovar Pomona in another seal dropped from 12,800 in May to <1/50 in June. These seals also had titres to serovar Hardjo, which rose or fell in the same manner. All suspicious or positive titres occurred in late April and early May, when the pups were approximately 4-5 months old. In June and July, all seals tested were negative. CONCLUSIONS: There was no serological evidence of Brucella infection in the pre-weaned fur seals at the colony. Positive titres to serovars Pomona, Hardjo, or Canicola suggest that a Leptospira species was present at the colony, however isolation or visualisation of the organism is required to confirm this. Care should be exercised when handling New Zealand fur seals to prevent human infection or inadvertent transfer of leptospirosis to another marine mammal species.     

A retrospective survey of the ocular histopathology of the pinniped eye with emphasis on corneal disease

Objective A retrospective review of globes from 70 pinnipeds submitted to the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) describing the type and frequency of ocular disease. Animals studied The study included 50 California sea lions, four animals listed only as ‘sea lion’, nine Northern elephant seals, five harbor seals, 1 Northern fur seal, and 1 Hooded seal. Procedures Globes were classified by microscopic findings. Categories were not mutually exclusive. Results The largest category was corneal disease (63 globes from 40 pinnipeds). The second largest was cataractous changes (35 globes from 23 pinnipeds). Additional ocular diseases included traumatic ocular injuries (nine globes from eight animals), phthisis bulbi (nine globes from eight pinnipeds), neoplasia (nine globes from six adult California sea lions), amyloid deposition in the corneal stroma, ciliary body, or both locations (five globes from four pinnipeds), and fungal disease (three globes from two pinnipeds). Pinnipeds with corneal disease were further categorized: stromal pathology (39 globes from 27 pinnipeds); epithelial pathology (37 globes from 27 pinnipeds); Descemet’s pathology (11 globes from eight pinnipeds); endothelial attenuation or absence (33 globes from 22 pinnipeds); presence of retrocorneal membranes (15 globes from 10 pinnipeds); anterior synechia (eight globes from six animals), and keratitis (seven globes from five pinnipeds). Conclusions This is the first report of ocular amyloid in pinniped eyes. All cases of neoplasia were in a pattern suggesting metastatic disease. In this study, there was a higher prevalence of ocular disease in captive pinnipeds, particularly in the posterior cornea.     

Polyunsaturated fatty acids and parasitism: effect of a diet supplemented with fish oil on the course of rat trichinellosis

Dietary fish oil has a beneficial effect on heart and some bacterial diseases and apart from other effects, some studies have revealed their ability to modulate the course of inflammatory and autoimmune diseases. The study here reported was designed to evaluate the possible influence of a fish oil supplement on the course of a Trichinella infection. Nutritional, parasitological and immunological parameters were analyzed. Two groups of 20 Wistar rats, one fed a standard diet and the other one a standard diet supplemented with fish oil, were infected with 1000 L1 larvae. Other two uninfected groups served as control. Results were as follows: fish oil diet intake and infection have, respectively, a positive and a negative effect on growth and food utilization. The negative effect is detected later in animals fed the fish oil diet. A reduction of 30.9 and 36.6% in the number of adult worms and L1 larvae, respectively, was observed in the fish oil group as compared to the standard diet group. Production of IFNgamma (Th1 response) and IL4 (Th2) response was measured in stimulated splenic cells. The fish oil diet increased both IFNgamma and IL4 levels. At 6 days after infection both IFNgamma and IL4 responses were detected, but at 36 days after infection only IL4 was detected in the standard group. The level of somatic and cuticular antibodies was not affected by the diet.     

Serological survey of pre-weaned New Zealand fur seals (Arctocephalus forsteri) for brucellosis and leptospirosis

AIM: To conduct a longitudinal serological survey for evidence of Brucella spp and Leptospira spp infection of pre-weaned New Zealand fur seals in a colony on the Otago Peninsula.

METHODS: Seal pups were repeatedly captured on a monthly basis from February through to July 2001. Pups were tagged at first capture and a blood sample was taken at each capture event. A total of 163 sera were collected from 118 seal pups. Where sufficient volume was collected, the sera were tested for leptospirosis using the microscopic agglutination test (MAT), and for brucellosis using the competitive enzyme-linked immunosorbent assay (ELISA) for Brucella abortus.

RESULTS: None of 128 sera from 101 seals tested positive to the ELISA for B. abortus. All tests for Leptospira interrogans serovars Grippotyphosa, Copenhageni, Bratislava and Leptospira borgpetersenii serovar Ballum were negative at a cut-off of <1/100 dilution. Positive or suspicious titres were found to L. interrogans serovars Canicola and Pomona and L. borgpetersenii serovar Hardjo. The highest titres (12,800) were found to serovar Pomona. The titre to serovar Pomona in one seal rose from <1/50 in March to 12,800 in April and was <1/50 when re-sampled in July. The titre to serovar Pomona in another seal dropped from 12,800 in May to <1/50 in June. These seals also had titres to serovar Hardjo, which rose or fell in the same manner. All suspicious or positive titres occurred in late April and early May, when the pups were approximately 4–5 months old. In June and July, all seals tested were negative.

CONCLUSIONS: There was no serological evidence of Brucella infection in the pre-weaned fur seals at the colony. Positive titres to serovars Pomona, Hardjo, or Canicola suggest that a Leptospira species was present at the colony, however isolation or visualisation of the organism is required to confirm this. Care should be exercised when handling New Zealand fur seals to prevent human infection or inadvertent transfer of leptospirosis to another marine mammal species.     

Infection of Japanese quail with Toxocara canis larvae and establishment of patent infection in pups

Migration and distribution of Toxocara canis larvae in the tissues of Japanese quail, infected orally with 5 X 10(3) infective eggs, and the establishment of patent infection from the quails to the definitive host, were studied. Larval yield at necropsy from various tissues and organs of quail varied from 4.72 to 7.54% of the infective eggs inoculated within the period 1-60 days post-infection (PI). The total number of larvae recovered on different days showed a gradual increase. The percent inoculum recovered at necropsy was highest on Day 60. Most of the larvae were found in the liver throughout the period and only a few migrated to other tissues, such as lung, heart, muscle and brain. The establishment of patent infections in the definitive host was studied by feeding the 15-day infected livers of Japanese quail (400 larvae) to Toxocara-free pups. Eggs first appeared in the faeces 38 days post-infection, the mean worm burden at necropsy was 87 and the percentage of infection established was 21.75%. Thus the role of Japanese quail as a paratenic host of T. canis is established.     

Pathogenesis of two strains of lion (Panthera leo) morbillivirus in ferrets (Mustela putorius furo)

Canine distemper virus (CDV) was previously considered to have a host range restricted to the canid family. In 1994, the virus was associated with sporadic outbreaks of distemper in captive felids. However, after severe mortality occurred in the Serengeti lions (Panthera leo), attention became focused on the pathogenesis of the virus and a concerted effort was made to identify the virus as CDV or a closely related feline morbillivirus. The present study was designed to explore the susceptibility of ferrets to challenge with two morbilliviruses isolated from lions and the protective effects of a modified-live mink distemper vaccine. Because mortality in ferrets infected with pathogenic CDV approaches 100%, the ferret was selected as a test animal. Two strains of lion morbillivirus were used as a challenge, A92-27/20 (California lion isolate) and A94-11/13 (Serengeti lion isolate). The two strains of lion morbillivirus were antigenically related to CDV (Rockborn strain), and ferrets were susceptible to both of the viruses when inoculated intraperitoneally. The inoculated ferrets were anorectic at 5-6 days postinoculation (PI), exhibited oculonasal discharge at 9-12 days PI, and became moribund at 12-22 days PI. Severe bilateral conjunctivitis was the typical clinical sign. Inclusion bodies characteristic of morbillivirus (eosinophilic, intranuclear, and intracytoplasmic) were distributed in many epithelial cells, including those of the skin, conjunctiva, gallbladder, liver, pancreas, stomach, trachea, lung, urinary bladder, and kidney. Virus was reisolated from selected lung tissues collected at necropsy and identified by CDV-specific immunofluorescence. Ferrets vaccinated with the mink distemper vaccine (Onderstepoort strain) were protected from challenge with the two lion strains, adding further support to the premise that the viruses are closely related to CDV.     

Experimental Encephalitozoonosis in the Blue Fox: Neonatal Exposure to the Parasite

Newborn and young pups up to the age of 15 days were exposed to E. cuniculi, either by keeping the pups in cages together with orally inoculated foster-mothers and their offspring, or by oral inoculation with E. cuniculi spores. A majority of pups appeared sero-positive to E. cuniculi with the india-ink immuno-reaction from 35 to 87 days post exposure; spores of E. cuniculi were detected in organs of some of the animals. The non-inoculated pups kept together with the orally inoculated pups became seropositive from 49 to 129 days after the oral inoculations. However, the exposure of newborn and young pups failed to induce clinical encephalitozoo-nosis, and when killed at the time of pelting the body weights and fur quality appeared to be within the normal range in all exposed foxes. No macroscopic lesions were detected in the various organs. Histologically focal interstitial nephritis occurred in the great majority of the seropositive animals. Meningoencephalitis was seen in some of the foxes, whereas slightly thickened walls of some arteries, mainly in the myocardium, were found in a few animals. The lesions of the brain and kidneys seem to be very similar to those seen in chronic cases of rabbit encephalitozoonosis. Polyarteritis nodosa and severe encephalitis and interstitial nephritis with extensive proliferations of plasma cells, which are almost constant findings in cases of clinically diseased foxes, were not detected in any of the subclinically infected animals. Various factors that might be of significance in the pathogenesis of the disease are discussed, and it is concluded that intrauterine infection of the pups via the transplacental route appears to be an essential supposition for the establishment of clinical fox encephalitozoonosis.     

Early Kinetics of Infectious Hematopoietic Necrosis Virus (IHNV) Infection in Rainbow Trout

Abstract

A series of experiments was carried out with infectious hematopoietic necrosis virus (IHNV; 193-110 isolate) in rainbow trout Oncorhynchus mykiss (weight, ～1.2 g) to determine the duration of the patent period and the timing of onset of the infectious periods. We first attempted to transmit IHNV to recipient fish from infected rainbow trout 2–3 d after they had been exposed. No infection transfer occurred despite high titers (10^4.79 to 10^4.91 plaque-forming units 5–8 d postexposure (dpe). To determine the number of secondary cases produced by one infectious individual, we exposed approximately 50 rainbow trout (weight, ～1.5 g) in each of seven replicate tanks to a donor fish that had been infected with virus by bath exposure 3 d earlier. The prevalence of infection in recipient fish rose from 0.84% at 2 dpe to 7.9% at 6 dpe. Maximum incidence (22 cases) occurred between 2 and 4 dpe. No disease-specific mortalities occurred in recipient fish during the experiment. The titer of virus in both recipient and donor fish increased from 2 to 4 dpe. There was a positive correlation between the level of infection among donors and prevalence values among recipient fish (r ² = 0.60). The level of challenge by one infectious fish under the conditions provided was enough for infection transfer from sick cohabitant to susceptible fish but was not enough for initiation of a full-scale epizootic among recipients.     

Growth Dynamics of White Muscle Fibres in Relation to Somatic Growth of Larvae of European Sea Bass (Dicentrarchus labrax, L.)

The body length and diameter of muscle fibres from the right hypaxial tail myomeres were measured in 100 sea bass larvae (Dicentrarchus labrax, L.) at ages of 40 to 90 days, from a fish farm in Croatia. The larvae were kept at a temperature of 19-20 degrees C and fed with standard feed for their respective ages. Body lengths of larvae 40-90 days old varied from 8 to 42 mm, increasing significantly in 10-day intervals (p < 0.001), except between days 50 and 60 when there was no increase in the body length. The measured diameters of muscle fibres ranged from 10 to 80 microm, the mean value increasing except between days 50 and 60. The fibre diameter in larvae aged up to 60 days was 10-40 microm, while that in larvae older than 70 days was 51-80 microm. In 90-day-old larvae there were no fibres of 10-20 microm in diameter; however, the number of fibers of 61-80 microm diameter increased. A statistically significant concordance was determined between the body length and fibre diameter of larvae 70 to 90 days old. Between days 50 and 60, arrested growth of the larval body and corresponding decrease in fibre diameter was observed. From day 45 until days 59, larvae were fed with Artemia nauplii and microparticles of formulated diets. From the day 60, minced fish was added to formulated diets. This change in diet probably resulted in the observed growth arrest.