www.elsevier.comrlocateranireprosci
Embryonic mortality and embryo–pathogen
interactions
G. Vanroose
), A. de Kruif
1, A. Van Soom
2Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, UniÕersity of Gent,
Salisburylaan 133, 9820 Merelbeke, Belgium
Abstract
Ž .
Embryonic mortality EM has a substantial impact on the fertility of domestic animals. Most of the embryonic losses occur during the first days after fertilization and during the process of implantation. Causes of EM can be divided into infectious and non-infectious categories. Primary attention has often been given to infectious agents but non-infectious causes probably account for 70% or more of the cases of embryonic death.
Infection of the embryonic environment can be caused by specific and non-specific uterine pathogens. Specific uterine infections are caused by a number of viruses, bacteria and protozoa that enter the uterus by the haematogenous route or via the vagina. Non-specific pathogens are mainly bacteria that enter the uterus by ascending infection. Uterine pathogens may cause EM by
Ž .
changing the uterine environment endometritis or by a direct cytolytic effect on the embryo. Ž
Non-infectious causes of EM such as chromosomal aberrations, external factors e.g., high
. Ž
ambient temperature and nutritional factors and maternal factors e.g., hormonal imbalances and .
age are multifactorial and difficult to diagnose.q2000 Elsevier Science B.V. All rights reserved.
Keywords: Embryonic mortality; Infection; Embryo–pathogen interaction
1. Introduction
Ž Ž . .
Prenatal losses embryonic mortality EM and fetal death are the most important causes of reproductive losses in animals and have a substantial impact on the profitabil-ity of the animal production.
)Corresponding author. Tel.:q32-9-264-75-61; fax:q32-9-264-75-63.
Ž . Ž .
E-mail addresses: geert.vanroose@rug.ac.be G. Vanroose , aart.dekruif@rug.ac.be A. de Kruif ,
Ž .
ann.vansoom@rug.ac.be A. Van Soom .
1
Tel.:q32-9-264-75-63
2
Fax:q32-9-264-77-97
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
Most of these losses occur during the embryonic period of gestation. This period Ž
extends from fertilization to the completion of the differentiation stage an embryo is .
considered a fetus when mineralization begins . Furthermore, most of the embryonic losses occur during the first days after fertilization and during the process of
implanta-Ž .
tion Wathes, 1992 . The adhesion stage of the implantation process in domestic animals starts at day 14 in sows, days 15–16 in ewes, day 16 in bitches, days 18–20 in goats,
Ž .
days 21–22 in cows, and days 36–40 in mares Gandolfi et al., 1992; Guillomot, 1995 . Ž
EM has been estimated to be about 20–40% in cows Lopez-Gatius et al., 1996; Hanzen
´
. Ž .
et al., 1999 ; 10–40% in sows Lambert et al., 1991; Gordon, 1997 , 10–30% in goats
Ž .
and 15–60% in mares Allen, 1992; Bergfelt and Ginther, 1992 . Fetal death has been
Ž . Ž
estimated to be about 5% Lambert et al., 1991 , but may exceed 10% Lopez-Gatius et
´
. al., 1996 .
Prenatal losses can be caused by infections and by non-infectious factors. Primary attention has often been directed to infections but non-infectious causes probably
Ž .
account for 70% or more of the cases Christianson, 1992 . Non-infectious causes are often multifactorial and are difficult to diagnose.
This paper aims to give a review of the causes of EM in domestic animals, and to focus upon some species-specific causes. Particular focus is concentrated on embryo– pathogen interactions in the discussion.
2. Infectious causes
Viral, bacterial, protozoal and, possibly, mycoplasmal infections can result in embry-onic death, indirectly by systemic effects via septicemias, viremias, or toxemias on the dam, or directly by affecting the embryo or contaminating its environment.
EM caused by systemic pathogens is usually related to fever during the infection. High fever present in the first stage of pregnancy can lead to early embryonic death as a result of denaturation of embryonic proteins. Prostaglandines, which may be elevated in febrile states, can cause luteolysis and subsequent loss of pregnancy. Furthermore, the stress condition present when an animal is febrile may indirectly lead to loss of pregnancy through the elevated steroids by themselves and through a lowered immune
Ž .
response to other organisms that can cause EM Christianson, 1992 .
Direct infection of the embryonic environment and of the embryo is another cause for EM.
2.1. Infection of the embryonic enÕironment
Ž .
Infection of the embryonic enÕironment oviduct and uterus can be caused by
specific and non-specific uterine pathogens. Specific uterine infections are caused by a number of viruses, bacteria and protozoa. These pathogens enter the uterus by the
Ž .
haematogenous route e.g., primary infection of the female with Toxoplasma gondii or
Ž . Ž
via the vagina at natural service e.g., Campylobacter fetus or at insemination e.g.,
Ž ..
pathogens are mainly bacteria that enter the uterus by ascending infection or at insemination. Sometimes they cause endometritis. The infection and the resulting inflammatory products must be eliminated before the embryo descends into the uterus.
Ž .
Particularly in older animals e.g., the mare , this uterine clearance can be impaired.
Acute endometritis, after mating or artificial insemination, has a direct effect on the
embryonic environment and is, in severe cases, accompanied by the production of
Ž .
luteolytic substances such as prostaglandines De Winter et al., 1995 . Bacterial uterine infections cause mostly a diffuse and severe purulent inflammation. Viral infections are, in most cases, characterized by a necrotizing endometritis, causing diffuse and total lymphocytic and plasmacytic changes in the endometrium.
Chronic endometritis involves a range of morphological and functional changes in
the uterus besides inflammation. The deposition of layers of fibrous tissue around endometrial glands results in a deficiency of functional glands. This deficiency will deprive the embryo of the protein-rich exocrine secretion.
2.2. Infections of the embryo
Infections of the embryo proper can take place at two important phases in the embryonic development: before hatching and after hatching.
( )
2.2.1. Before hatching zona pellucida-intact embryos
Of all pathogens, viruses are the most insidious and dangerous type of infection for the early-stage embryo. Viral infection of the zona pellucida-intact embryo can already have taken place before fertilization during maturation of the oocyte. Viruses, e.g.
Ž .
bovine herpes virus 1 BHV-1 and BVDV, might be present in follicular fluid or
Ž .
granulosa cells of bovine oocytes Bielanski et al., 1993 , and can also contaminate the embryos by adhering to the glycoprotein layer, which surrounds the oocyte, the so-called zona pellucida. In persistently-infected cattle, BVDV antigen has even been detected
Ž .
inside the oocyte Brownlie et al., 1997 . Virus adhering to the zona pellucida or to the fertilizing spermatozoon might be introduced into the oocyte by the sperm track in the
Ž .
zona pellucida created at the time of fertilization Bowen, 1979 . Passive migration of virus through the meshes in the zona pellucida is highly unlikely to occur, since particles
Ž .
with a diameter of 40 and 200mm comparable size as BVDV and BHV1 remain stuck
Ž .
in the peripheral part of the zona pellucida Vanroose, 1999 . Only one report has ever shown that one of the smallest viruses, the porcine parvovirus, could pass the zona
Ž .
pellucida in pigs Bolin et al., 1983 . After fertilization, the zona pellucida can be Ž
considered as an effective barrier for virus penetration Stringfellow et al., 1991; .
Vanroose et al., 1999a . However, at these early embryonic stages, death of a zona pellucida-intact embryo can occur because of a hostile uterine environment.
2.2.2. After hatching
Ž .
agents Wrathall and Sutmoller, 1998 . For example, zona pellucida-free bovine morulae
¨
Ž
and blastocysts are susceptible to bovine herpesvirus-1 Bowen et al., 1985; Bielanski et
. Ž
al., 1987; Vanroose et al., 1997 and BVDV Brock and Stringfellow, 1993; Vanroose et
. Ž .
al., 1998 , but not to bovine parvovirus Bowen, 1979 . Recent research on embryo– pathogen interactions has mainly been performed in cattle, by exposing in vivo derived
Ž
or in vitro produced embryos to specific pathogens in vitro Wrathall and Sutmoller,
¨
. 1998; Vanroose, 1999 .
Besides these in vitro studies, earlier in vivo studies have shown that viral uterine infections can result in extensive viral replication in embryonic cells after hatching and implantation. In addition, cells undergoing rapid division, as occurs in embryos, are
Ž .
particularly susceptible to the replication of certain viruses Bowen, 1979 . The outcome of such an infection can either be cytolytic or non-cytolytic. Both can result in EM, but a non-cytolytic infection can also cause chromosomal damage and induce embryonic cells to divide more slowly. This retardation in cell division occurs during the critical phase of organogenesis. Consequently, viral infection of embryos may result in the develop-ment of congenital malformations.
After implantation, the haematogenous route of uterine infection becomes more important, since both endometritis and a direct cytolytic effect on the embryo are possible.
3. Non-infectious causes
3.1. Chromosomal aberrations
Chromosomal aberrations are a major cause of early pregnancy failure in animals. ŽKing, 1990 . A range of misalliances can occur during the pairing of the haploid. parental chromosome sets at the time of fertilization, which are subsequently lethal to the embryo. Chromosomal abnormalities may also originate by penetration of more than
Ž .
one sperm cell polyspermia . Mixoploidy, polyploidy and haploidy are all aberrations Ž
that are encountered frequently in in vitro produced embryos Kawarsky et al., 1996; .
Viuff et al., 1999 but it has not been investigated yet whether this could be a cause for the higher EM rates, which are observed after the transfer of in vitro-produced bovine
Ž . Ž .
embryos Van Soom et al., 1994 . King 1990 reported that chromosomal abnormalities may account for approximately 20% of the total embryonic and fetal loss. Sometimes an enzyme deficiency results in early EM, e.g. deficiency of uridine monophosphate
Ž . Ž
synthetase DUMPS in cattle, which is an autosomal recessive disorder Schwenger et .
al., 1993 .
3.2. External factors
3.2.1. High enÕironmental temperatures
High environmental temperatures during the first months of gestation can also have
Ž .
embryo and by the shunting of blood away from the uterus to the periphery in an
Ž .
attempt to maintain body temperature, resulting in a reduced nutrient load Dziuk, 1992 .
3.2.2. Specific nutrient deficiencies or malnutrition
Specific nutrient deficiencies or malnutrition can have a negative effect on the
Ž . Ž
embryo. Especially a severe deficiency of vitamins vitamin A or other nutrients Cu,
. Ž .
Zn, I that serve as regulators of metabolism can cause EM Graham et al., 1995 . Malnutrition or a severe negative energy balance may affect the follicular development, the quality of the oocyte, and the secretory and motile activity of the oviduct which is the place of the fertilization process. These findings demonstrate that nutrition affects
Ž .
the very early stages of conceptus Butler and Smith, 1989; Foxcroft, 1997 .
3.2.3. Stress
Ž
Stress has a deleterious effect on reproductive efficiency in animals Dobson and
. Ž
Smith, 1995 . Stressors e.g., transport, mechanical injury, isolation, pain, changes in .
blood pressure, . . . affect the reproductive function via actions at the hypothalamic
Ž . Ž .
level GnRH or at the ovarian level progesterone .
3.2.4. EnÕironmental toxicants, teratogenic compounds and mycotoxins
Environmental toxicants, teratogenic compounds and mycotoxins can have drastic adverse effects on the survival of embryos when ingested at crucial early stages of
Ž .
gestation Christianson, 1992; Brendemuehl et al., 1994 .
3.3. Maternal factors
3.3.1. Hormonal imbalance
Progesterone is necessary for the maintenance of pregnancy. A deficiency of proges-terone caused by primary luteal insufficiency has been reported as a cause of EM, but is
Ž .
probably not of frequent occurrence Wathes, 1992; Mann et al., 1998 .
3.3.2. Disturbance of the embryo–maternal interactions
Disturbance of the embryo–maternal interactions can result in embryonic loss. Before implantation, embryonic signalling is necessary for maternal recognition of pregnancy. This initiates the hormonal changes, which are needed to elicit the uterine
transforma-Ž
tions necessary for implantation Gandolfi et al., 1992; Geisert et al., 1992; Hansen, .
1997 .
3.3.3. Insufficient uterine space
Ž .
Insufficient uterine space results in embryonic loss Allen, 1992; Dziuk, 1992 . The presence of more than one embryo in cattle and horses and more than 20–25 embryos in swine causes a competition between the embryos for access to the endometrium. For
Ž
example, in the mare, natural embryo reduction of twin embryos 10–20% of the .
pregnancies occurs in a majority of the cases, especially between the second and sixth
Ž .
3.3.4. Age of dam
Older animals have lower follicular activity and lower oocyte quality resulting in a decrease of the developmental competence of embryos. Furthermore, the quality of the endometrium is deteriorating with increasing age.
3.3.5. Inbreeding
Ž .
Inbreeding has been reported as a cause of EM Hanzen et al., 1999 . It has also been proven that the EM differs from breed to breed.
4. Species-specific causes of embryonic death
4.1. Cattle
4.1.1. Infectious causes
Many viruses, bacteria and protozoa can be associated with EM and early fetal loss. Bluetongue virus, BVDV and BHV-1 are the most important viral agents. From in vivo experiments with in vivo-derived embryos, it is known that BHV-1 causes infection
Ž .
in hatched embryos that result in EM Miller, 1991 . Furthermore, it is known that, particularly in BHV-1-seronegative cattle, artificial insemination with BHV-1-con-taminated semen can result in markedly reduced conception rates and endometritis. The outcome of infection depends on the amount of virus per straw and the properties of the virus strain. Natural breeding with bulls shedding BHV-1 in their semen does not appear
Ž .
to affect fertility van Oirschot, 1995 . The in vitro infection of zona pellucida-intact in vivo-derived embryos has no effect on the embryonic development. However, the exposure of hatched in vivo-derived embryos to BHV-1 was rapidly embryocidal ŽBowen et al., 1985; Bielanski et al., 1987; Wrathall and Sutmoller, 1998 .
¨
.Infections of BVDV-seronegative heifers with BVDV resulted in a lower pregnancy rate. Artificial insemination or natural breeding with BVDV-contaminated semen can result in fertilization failure or embryonic death. The incubation of zona pellucida-intact in vivo derived embryos with BVDV has no effect on embryonic survival, but the exposure of hatched embryos to cytopathic BVDV causes EM.
Recently, the interactions of BHV-1 and BVDV with in vitro-produced embryos were investigated by experimental in vitro infections. It was demonstrated that embryonic cells of early zona pellucida-free oocytes and zygotes were refractory to an infection
Ž .
with BHV-1 and BVDV Vanroose et al., 1997, 1998 . In more advanced stages, such as zona pellucida-free 8-cell stage embryos, zona pellucida-free morulae and hatched blastocysts, newly produced BHV-1 or BVDV was detected. It was observed that the exposure of zona pellucida-free embryos to BHV-1 and to a cytopathic BVDV strain results in destruction of the embryonic cells. Furthermore, both viruses could replicate in
Ž oviductal cells, resulting in cell lysis and elimination of their biological functions such
.
as secretion of embryotrophic factors supporting embryonic development . The presence of BHV-1 and BVDV in an in vitro embryo production system has clear adverse effects
Ž
on fertilization and embryonic development Guerin et al., 1992; Bielanski and Dubuc,
´
.
demonstrated that the zona pellucida of in vitro-produced embryos was an effective barrier against viral infections, but the outer pores of the zona were large enough to
Ž
allow entry of BHV-1 and BVDV in the outer layers of the zona pellucida Vanroose, .
1999 . Consequently, the embryo can become infected when it hatches out of a virus-contaminated zona pellucida. The importance of the zona pellucida was also
Ž
demonstrated for in vivo-derived embryos Singh et al., 1982a, 1982b; Potter et al., . 1984; Bielanski and Hare, 1988; Gillespie et al., 1990; Stringfellow et al., 1991 .
Some bacterial and protozoal infections, such as trichomoniasis and campylobacterio-sis, which are venereal diseases, are characterized by endometritis resulting in infertility and EM. Other infections such as brucellosis, Arcanobacter pyogenes infection, can-didiasis, leptospirosis, neosporosis, fungal infection, listeriosis, and Haemophilus
som-Ž
nus infection are more associated with late EM, fetal death and abortion Larsson et al.,
. 1994; Sekoni, 1994; McGowan and Kirkland, 1995; Moen et al., 1998 .
4.1.2. Hormonal imbalances
A number of studies have demonstrated a relationship between low maternal proges-terone levels and early pregnancy failure. Both a late post-ovulatory progesproges-terone rise and low luteal phase concentrations are associated with poor embryo development and the production of insufficient interferon-t to prevent luteal regression. The
post-ovula-tory rise of progesterone is of particular interest as it maintains the synchrony between embryo and uterus. An overall analysis of studies concerning progesterone supplementa-tion revealed a significant improvement in the pregnancy rate of 5% following
proges-Ž .
terone supplementation Mann et al., 1998 .
4.1.3. Trauma
Trauma after pregnancy diagnosis by rectal palpation or by using an ultrasound scanner can result in pregnancy loss. In cattle, early pregnancy diagnosis is generally performed between 35 and 50 days of gestation, which is the period of the completion of the differentiation. Therefore, the risk exists that the embryo or the fetus becomes
Ž .
damaged. However, Vaillancourt et al. 1979 found no indication that embryonic loss at the time of or shortly after early pregnancy examination was increased. Recently, Baxter
Ž .
and Ward 1997 reported that ultrasound examination has no detrimental effect on the fetus. Rectal palpation is also a safe procedure when performed correctly.
4.1.4. NegatiÕe energy balance
A severe negative energy balance of high yielding dairy cows after calving may Ž affect oocyte quality and may enhance EM once the cow has been inseminated Butler
. and Smith, 1989 .
4.2. Horse
4.2.1. Infectious causes
A large number of bacteria can infect the endometrium resulting in failure of fertilization or EM. They are often opportunist pathogens that can be isolated from the genital tract of normal mares, i.e. E. coli and Streptococcus spp. Others are considered to be venereal pathogens, i.e. Pseudomonas spp., Klebsiella spp. and Taylorella
Ž .
equigenitalis contagious equine metritis .
4.2.2. Persistent endometritis, chronic degeneratiÕe endometritis and endometriosis
Persistent endometritis, chronic degenerative endometritis and endometriosis are major causes of reduced fertility in brood mares. These are age- and parity-related. A persistent inflammation of the uterus results in premature luteolysis and EM in response to increased PGF2aconcentrations. The inflammation can also interfere with the survival of an embryo. After fertilization has taken place the embryo remains in the oviduct for 5–6 days. The embryo then descends into the uterine lumen where the presence of fluid, bacteria, and inflammatory products is incompatible with its survival. Chronic degenera-tive endometritis and endometriosis involve a range of morphological and functional changes in the uterus such as periglandular fibrosis of the uterine glands and a deficiency of functional glands. The uterine glands produce ‘uterine milk,’ which is, until day 40, the only source of nutrition available to the still unimplanted conceptus. Thus, deficiency in nutrients will affect the fetus, right up to the point of starvation and
Ž .
death Allen, 1992 . Endometriosis is also associated with the development of large lymph-filled endometrial cysts that protrude in the uterine lumen. Cysts have no microcotyledons, and therefore, there is a proportional reduction in the nutritional sustenance of the embryo. In addition, large cysts or a large quantity of small ones can prevent the migration of the conceptus during the critical period between days 12 and 16
Ž
after ovulation resulting in a failure of the maternal recognition of pregnancy Thatcher .
et al., 1997 . Finally, chronic degenerative endometritis and endometriosis may cause Ž
early EM by the loss of myometrial tone Nikolakopoulos and Watson, 1999; Troedsson, .
1999 . These features result in inadequate expulsion of endometrial gland secretions of oestrus, together with seminal fluid, penile smegma, and bacteria before the cervix contracts following ovulation. Consequently, when the embryo reaches the uterus, it
Ž .
enters a mixture of debris and it quickly succumbs Allen, 1992 .
4.2.3. Chromosomal abnormalities
Chromosomal abnormalities are a major cause of early pregnancy failure in horses. These failures may start very early but the majority were seen to occur between 20 and 30 days of gestation, which is the period when the embryo is undergoing all the changes
Ž .
associated with organogenesis Allen, 1992 .
4.2.4. Hormonal deficiencies and imbalance
Ž
Progesterone is critical for the maintenance of pregnancy in mares Daels et al., .
1991 . The only source of progesterone during the embryonic period is the primary Ž corpus luteum. Primary luteal insufficiency is a cause of early embryonic death Pycock
.
and Newcombe, 1996 , but is of rare occurrence.
4.2.5. Twin pregnancy
4.2.6. Stress
Stress due to malnutrition, transport and severe pain has been implicated as a cause of EM. However, the relationship between malnutrition and embryonic death has not been proven. Also, transport of pregnant mares did not result in a lower pregnancy rate. On the other hand, severe pain, for example caused by colic can result in luteolysis and thus
Ž .
EM Daels et al., 1991 .
4.3. Swine
4.3.1. Infectious causes
Infections play an important role in prenatal losses in swine and can be categorised based on whether the agents exert systemic effects, e.g. swine influenza virus, or infect the embryo directly, e.g. pseudo-rabies virus. Infections before 35 days lead to
embry-Ž
onic resorption or early abortion. Viral infections e.g., porcine enteroviruses, porcine .
parvovirus, pseudo-rabies virus, or classical swine fever after 35 days will often result
Ž .
in mummified fetuses Christianson, 1992 .
Many ubiquitous bacteria can cause endometritis and as a result embryonic death, for example E. coli, Erysipelothrix rhusiopathiae, Listeria spp. and Staphylococcus spp.
Ž .
De Winter et al. 1995 have demonstrated that the syndrome of endometritis post-in-semination can be caused by ascending infections with facultatively pathogenic bacteria present in the vagina or in the semen. Such infections do not impair fertilization but disturb the embryo–maternal interactions or disrupt the process of implantation of the embryos. This results in vaginal discharge 14–25 days after insemination. When the uterus has already been infected before service, e.g. because of a chronic infection after a previous insemination the uterine environment has changed very much. As a conse-quence, fertilization will not take place or early embryonic development is disturbed
Ž .
resulting in EM before day 11 De Winter, 1995 .
The sow’s endometrium has the best resistance to these uterine infections during oestrus, but is already susceptible to bacterial infections at the end of oestrus.
4.3.2. Number of embryos
Ž .
In swine, at least four embryos two in each horn are needed at the time of
Ž .
implantation for maintenance of pregnancy Christianson, 1992 .
4.3.3. Variation in deÕelopment
Twelve days after insemination, there is considerable variation in morphological
Ž .
development between littermates Lambert et al., 1991 . It has been postulated that the more-developed embryos within the litter advance uterine secretions by synthesizing more oestradiol than their lesser-developed littermates. As a result, the lesser-developed embryos probably become more susceptible to this asynchronous environment and consequently die.
4.3.4. Stress
Stress due to malnutrition and transport of pregnant sows has been implicated as a
Ž .
4.3.5. High plane of nutrition
The high plane of nutrition early in pregnancy can have an adverse effect on embryo survival. A very high level of dietary protein induces a greater activity of enzymes, resulting in an increased rate of metabolism causing a reduction of progesterone that is
Ž .
needed for the embryonic development Dziuk, 1992 .
4.3.6. Season infertility
A reduction in the fertility in pigs in the summer and early autumn has been reported in many countries and appears to manifest as a range of problems from silent oestrus and ovarian cysts to EM.
4.4. Dog and cat
4.4.1. Infectious causes
The premature termination of gestation by embryonic or early fetal death is uncom-mon in the bitch. Minute virus of canines may cause transplacental infections with
Ž .
embryo resorptions Carmichael et al., 1991 . In cats, feline leukaemia virus can cause embryonic resorption. Other viruses causing EM and fetal death are feline panleucopenia virus, feline infectious peritonitis virus and feline herpesvirus 1.
4.4.2. Habitual foetal death
Some bitches and queens have a true luteal insufficiency resulting in habitual
Ž .
resorption of the conceptuses Okkens et al., 1992; Roth et al., 1995 . Particularly in the bitch, EM can result in the diagnosis of pseudo-pregnancy. In the queen, approximately 30% of all ovulated oocytes are either not fertilized or undergo pre-implantation EM ŽSwanson et al., 1994 ..
4.4.3. Cystic endometrial hyperplasia in the bitch and queen
Cystic endometrial hyperplasia, which precedes pyometra, may also result in concep-Ž
tion failure, failure of implantation and embryonic resorption Okkens et al., 1992; Roth .
et al., 1995 .
5. Conclusions
EM has a substantial impact on the fertility of domestic animals. Infectious causes are, perhaps, overemphasized but are certainly important in epidemic situations. Infec-tions can cause EM by changing the embryonic environment, by inflammation of the
Ž
endometrium, by a direct effect on the embryo and by systemic effects fever and .
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