Identification of a bovine enteric syncytial virus as a nongroup A rotavirus

An atypical or nongroup A rotavirus was identified in feces obtained from gnotobiotic calves in which fecal preparations originally derived during an epizootic of neonatal calf diarrhea had been serially passaged. The epizootic was previously reported to be caused by a noncharacterized viral agent that induced the formation of epithelial syncytia on small intestinal villi of experimentally infected calves. This bovine, nongroup A rotavirus was found to be antigenically related to a described atypical rotavirus of rats by immunofluorescence and by enzyme immunoassay. Complementary DNA derived from the atypical rat rotavirus cross hybridized with RNA obtained from the bovine virus, but not with RNA extracted from group A rotaviruses. Complementary DNA derived from SA-11 group A rotavirus cross hybridized with other group A rotavirus RNA, but not with RNA obtained from either the rat or bovine nongroup A isolates. Additionally, another similar, if not identical, bovine atypical rotavirus was identified in a second epizootic of neonatal calf diarrhea that occurred several hundred kilometers and 8 months apart from the original epizootic.     

Intestinal antibody response after vaccination and infection with rotavirus of calves fed colostrum with or without rotavirus antibody

The intestinal and systemic antibody response of calves vaccinated and/or challenged with rotavirus was studied employing isotype-specific ELISAs for the detection of IgG1, IgG2, IgM and IgA antibodies to rotavirus. Monoclonal antibodies to bovine immunoglobulin isotypes of proven specificity were used as conjugated or catching antibody. Five days after oral inoculation (dpi) of a 5-day-old gnotobiotic calf with rotavirus, IgM rotavirus antibodies were excreted in faeces, followed 5 days later by IgA rotavirus antibodies. The increase in IgM rotavirus antibody titre coincided with the inability to detect further rotavirus excretion. Faeces IgM and IgA rotavirus antibody titres fell to low levels within 3 weeks post infection. IgG1 and IgG2 rotavirus antibodies were not detected in faecal samples. In serum, antibodies to rotavirus of all four isotypes were detected, starting with IgM at 5 dpi. Two SPF-calves, which were fed colostrum free of rotavirus antibodies, were vaccinated with a modified live rotavirus vaccine and challenged with virulent rotavirus 6 days later. Upon vaccination, the calves showed an antibody response similar to the response of the infected gnotobiotic calf. Intestinal IgM rotavirus antibodies were excreted before or on the day of challenge and appeared to be associated with protection against challenge infection with virulent virus and rotavirus-induced diarrhoea. In 3 control calves, which were challenged only, the antibody patterns also resembled that of the gnotobiotic calf and again the appearance of IgM rotavirus antibodies coincided with the end of the rotavirus detection period. Two other groups of 3 SPF-calves were treated similarly, but the calves were fed colostrum with rotavirus antibodies during the first 48 h of life. These calves excreted passively acquired IgG1 and IgG2 rotavirus antibodies in their faeces from 2 to 6 days after birth. After vaccination, no IgM or IgA antibody activity in serum or faeces was detectable. Upon challenge, all calves developed diarrhoea and excreted rotavirus. Seven to 10 days after challenge low levels of IgM rotavirus antibody were detected for a short period. These data indicate that the intestinal antibody response of young calves to an enteric viral infection is associated with the excretion of IgM antibodies, immediately followed by IgA antibodies. This response is absent or diminished in calves with passively acquired specific antibodies which may explain the failure to induce a protective intestinal immune response by oral vaccination with modified live rotavirus of calves fed colostrum containing rotavirus antibodies.     

Isolation and characteristics of an equine reovirus type 3 and an antibody prevalence survey to reoviruses in horses located in New York State

Reoviruses have been isolated from a number of species including human, bovine, feline, canine and equine. In most species they seem to produce mild to inapparent disease. We have isolated a reovirus type 3 from a foal with diarrhea. The virus designated the Ralph strain has been propagated in both the MA-104 and A-72 cell lines. The strain produced cytoplasmic inclusion bodies in these cell cultures. Tissue-cultured virus fixed complement in the presence of reovirus antibodies, but failed to do so in the presence of rotavirus antiserum. By electron microscopy the viral particle measured +/- 65 nm. The virus hemagglutinated pig erythrocytes, but not human O, human A, calf, cow, chicken or guinea pig erythrocytes. In the hemagglutination test there was complete reciprocal crossing between the Ralph strain and the NIH reovirus type 3, but there was no crossing with the NIH reovirus types 1 and 2. A limited serological survey was completed on serum samples from New York State horses collected in 1976-1977 and 1981 using the hemagglutination-inhibition test. The percentage with antibodies to reovirus types 1, 2 and 3 for 1976-1977 was 24.5, 42.2 and 3.9% and in 1981, 8.8, 9.8 and 3.9%, respectively.     

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.     

Diagnosis of viral agents associated with neonatal calf diarrhea.

During this study, 134 samples have been examined for the detection of the viruses associated with neonatal calf diarrhea. The presence of Nebraska viruses (rotavirus and coronavirus) has been demonstrated by using the electron microscope and the fluorescent antibody techniques while the presence of other viruses has been detected by the observation of a cytopathic effect on monolayer cells of calf testis. The Nebraska viruses have been demonstrated in 107 (80%) out of 134 field case specimens. An association of rotaviruses and coronaviruses was found in 58 cases (54%) whilst the coronaviruses and the rotavirus were found singly in 34 cases (53%) and in 15 cases (14%) respectively. Four bovine virus diarrhea viruses, two infectious bovine rhinotracheitis viruses and two enteroviruses have also been isolated in the preceding 107 Nebraska positive specimens. For the detection of the Nebraska viruses, the fluorescent antibody techniques were more sensitive than the electron microscopy. However, those two techniques must be used simultaneously for a better detection of a greatest possible number of cases.     

Efficacy of an orally administered modified-live porcine-origin rotavirus vaccine against postweaning diarrhea in pigs

A commercially available, porcine-origin rotavirus vaccine was evaluated for efficacy against postweaning diarrhea due to rotavirus infection in pigs. Weight gains were compared at 5 intervals after weaning. Visual scoring was used to evaluate fecal consistency. Rectal swab specimens were cultured for hemolytic E coli and evaluated for rotaviral antigen by use of enzyme-linked immunosorbent assay. Milk from dams and sera from pigs and dams were evaluated for rotavirus-neutralizing antibodies by use of a plaque-reduction test. Significant differences between vaccinates and controls were not found in the determinants evaluated. Selected rotavirus-positive fecal and rectal swab specimens were examined for double-stranded (ds) RNA by use of direct electropherotyping, and the results were compared with the dsRNA pattern of rotavirus propagated from the vaccine. Only electropherotypes typical of field strain virus were found in the fecal and rectal swab specimens evaluated. Sera from guinea pigs and from a gnotobiotic pig immunized against the field strain rotavirus neutralized Ohio State University strain rotavirus (homologous to the vaccine rotavirus strain), but did not neutralize the Gottfried strain of rotavirus. This indicated that, although the dsRNA electropherotypes of the field and vaccine strains of the virus were different, the serotypes were similar, if not identical.     

Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea.

Ninety-one cases of neonatal calf diarrhea were examined for viruses with negative contrast electron microscopy. Viruses were demonstrated in 41% of the cases. Reo-like viruses and corona-like viruses, and mixed virus populations were observed in 12%, 20% and 9%, respectively. Twenty-six of the cases were examined by negative contrast electron microscopy, and by virus isolation or by fluorescent antibody technique. There was an 81% agreement in obtained results. The disagreements resulted from the demonstration of a viral agent by negative contrast electron microscopy while the other techniques did not indicate a virus. The results suggest that negative contrast electron microscopy is a more sensitive diagnostic tool for demonstration of viruses associated with neonatal calf diarrhea than are viral isolation or the fluorescent antibody technique.     

Sequential isolation of rotavirus from individual calves

An outbreak of neonatal calf diarrhea was studied on a breeding farm of Japanese indigenous beef cattle. During the breeding season of 1982, 43 calves were born over the period 27 February-28 April. All but one of the calves suffered from neonatal diarrhea and 5 died. Bovine rotavirus was isolated in cell cultures from fecal specimens of 39 (90.7%) of the 43 calves during the outbreak, strongly suggesting that this was the causative agent; the virus was readily isolated from 81 (83.5%) of 97 specimens of diarrhea. Rotavirus was subsequently isolated from the feces of 7 of the calves in early May, more than one month after the initial virus isolation in early March. Two of these calves were again rotavirus-positive in early June, 41 days after the second virus isolation. Diarrhea had ceased in all 7 calves in March. Some antigenic differences were shown by the neutralization test between the early and later isolates from one of these calves, suggesting either re-infection with a serologically different virus, or persistent infection with the original virus following antigenic modulation.     

Bovine Fetal Inoculations with Calf Rotavirus

The serological and histopathological responses of bovine fetuses to in utero inoculation with virulent and attenuated strains of the calf rotavirus (reovirus-like agent of neonatal calf diarrhea) are described. Thirteen bovine fetuses, 63 to 190 days of gestation, were inoculated in utero with attenuated (three fetuses) or field strain virus (nine fetuses) or both (one fetus).

Serum-neutralizing antibody titers ranging from 1:16 to > 1:256 were detected in six of eight fetuses tested, demonstrating the ability of the bovine fetus to respond immunologically to this agent. The youngest fetus in the series was inoculated at 63 days of gestation and developed a titer of 128 in 64 days. This represents the earliest stage of gestation at which a bovine fetus has been inoculated with a bovine virus and found to produce antibody to it. Serum neutralizing titers in six of the eight dams tested increased significantly following the inoculations of their fetuses in utero.

Histological changes associated with viral replication and antigenic stimulation of the lymphoreticular system were observed. Pneumonic lesions consisting of both local and diffuse lymphoreticular proliferation were present in five of the nine fetuses that were alive at slaughter. Gliosis and perivascular cuffing were noted in the brains of two of these fetuses and meningitis was seen in one. No evidence of teratogenic change was found.

     

Neonatal calf diarrhea induced by rotavirus

This presentation summarizes the results of a comprehensive study on rotaviruses isolated in Italy from calves and rabbits affected by neonatal diarrhea. The results clearly indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. The evidence of differences in virulence among bovine rotaviruses appeared also to be confirmed. Conventionally reared calves were fully susceptible to the experimental infection induced by three rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits, respectively. When rotavirus strains of bovine, simian, porcine and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain. On the other hand, a reciprocal antigenic correlation was found between bovine and rabbit isolates. Finally, it was proven that feeding newborn calves with colostrum of their dams, previously vaccinated with an inactivated rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

A CELL CULTURE VACCINE AGAINST BOVINE EPHEMERAL FEVER

SUMMARY A vaccine was prepared from cell culture fluids harvested from the twelfth passage of the 919 strain of bovine ephemeral fever (BEF) virus in Vero cell cultures. Cattle were vaccinated subcutaneously with various combinations of strain 919 virus and adjuvants. Neutralising antibodies were assayed at various times after vaccination and some cattle were challenged by intravenous inoculation with the virulent 417WBC strain of BEF virus. Strain 919 virus of the third and twelfth passage levels in Vero cells produced neither fever, clinical illness nor detectable viraemia in 5 calves inoculated intravenously. Nor could viraemia be detected in 5 heifers receiving vaccine subcutaneously. When the vaccine was administered mixed with aluminium hydroxide adjuvant, the production of neutralising antibodies increased with an increase in the volume of vaccine from 2.5 ml to 10 ml and the response to 2 injections was significantly better than the response to a single injection. The neutralising antibody response was decreased when vaccine was diluted in phosphate buffered saline. The neutralising antibody response following 2 subcutaneous vaccinations with strain 919 virus mixed with aluminium hydroxide adjuvant was higher than that following intravenous inoculation with virulent virus. The vaccine-induced antibodies persisted for at least 12 months, and revaccination at this time led to an increase in the titre of neutralising antibody. Antibodies induced by a single subcutaneous administration of strain 919 virus mixed with Freund's complete adjuvant persisted for at least 40 weeks; those induced by vaccine containing Freund's incomplete adjuvant had virtually disappeared within 16 weeks. All these calves responded to vaccination with aluminium hydroxide-containing vaccine with increases in levels of neutralising antibodies. Of 26 vaccinated calves challenged with virulent BEF virus, 24 remained clinically normal. Two developed brief periods of pyrexia on the seventh day after challenge, but no other clinical signs. One of these calves had a viraemia that was demonstrated only by intravenous inoculation of a susceptible calf. The remaining calf had no detectable viraemia. All of 7 unvaccinated calves developed severe clinical BEF within 5 days of challenge. No disease attributable to the 919 virus occurred in 24 vaccinated pregnant heifers or their newborn calves.     

一例冠状病毒、轮状病毒混合感染引起犊牛腹泻的诊治

2022年3月,河南省某规模化奶牛养殖场发生一起新生犊牛腹泻病例,在流行病学调查、临床症状观察、病理剖检的基础上,采集6头腹泻犊牛的新鲜粪便进行了寄生虫卵检查、小球隐孢子虫、牛轮状病毒、冠状病毒、大肠杆菌K99抗体检测,血液样品进行了牛病毒性腹泻病毒抗原检测,组织样品进行了细菌分离。结果表明牛轮状病毒抗原6头阳性,阳性率100%;冠状病毒抗原4头阳性、2头阴性,阳性率66.7%;其他病原均为阴性。根据临床症状、病理变化和实验室检测结果,确诊该病例为牛冠状病毒和轮状病毒混合感染引起的犊牛腹泻。根据诊断结果,采取了改善饲养管理、补液、收敛、止泻等综合性防治措施,疫情得到了有效的控制,为临床防治犊牛腹泻提供了参考。     

Immune electron microscopy of transmissible gastroenteritis virus and rotavirus (reovirus-like agent) of swine.

Immune electron microscopy (IEM) was developed as a diagnostic aid for detecting and identifying transmissible gastroenteritis virus and rotavirus (reovirus-like agent) in fecal and intestinal contents from cases of gastroenteritis in young pigs. Variables involved in use of direct IEM and its sensitivity were determined. Aggregates of virus coated with specific antibody were seen in virus samples mixed with homologous convalescent antiserum, but not in control samples containing preexposure serum or antibody directed against a heterologous virus. At least a ten fold enhancement of the sensitivity of direct IEM for virus detection was accomplished using indirect IEM employing rabbit anti-porcine IgG to further aggregate virus-antibody complexes. The technique was used to investigate the size and morphology of the porcine rotavirus. Particles ranged from 55 to 70 nm in diameter and had capsomere structures. Morphologically, the porcine rotavirus resembled the calf and human rotaviruses. By IEM, employing specific antiserums for each virus, porcine rotavirus was found to be antigenically related to these 2 viruses, but not to the reovirus type 3.     

A study on neonatal calf diarrhea induced by rotavirus

This review summarizes the results of a study on rotaviruses isolated from calves affected by neonatal diarrhea.

The results indicated that rotavirus infection is widespread and supported the evidence for an etiologic role of these viruses in neonatal diarrhea. Differences in virulence among bovine rotaviruses appeared also to be confirmed.

Conventionally reared calves were fully susceptible to the experimental infection induced by rotaviruses originating from heterologous hosts, i.e. monkeys, pigs and rabbits.

When rotavirus strains of bovine, simian and rabbit origin were compared by cross neutralization tests, it was found the simian and porcine strains were indistinguishable and both appeared to relate antigenically to the bovine strain.

Finally, it was proven that feeding newborn calves with colostrum and first milk of their dams, previously vaccinated with an inactivated adjuvanted rotavirus vaccine, could prevent the neonatal diarrhea from occurring.     

Induction of mucosal immune responses and protection against enteric viruses: rotavirus infection of gnotobiotic pigs as a model

Enteric viruses are a major cause of diarrhea in animals and humans. Among them, rotaviruses are one of the most important causes of diarrhea in young animals and human infants. A lack of understanding of mechanisms to induce intestinal immunity and the correlates of protective immunity in neonates has impaired development of safe and effective vaccines against enteric viruses. Studies of candidate vaccines using an adult mouse model of subclinical enteric viral infections often do not predict vaccine efficacy against disease evaluated in neonatal large animals. A series of studies have been conducted using a neonatal gnotobiotic pig model of rotavirus infection and diarrhea to identify correlates of protective immunity and to evaluate traditional and novel vaccine approaches for the induction of mucosal immune responses and protection to enteric viruses. Gnotobiotic pigs recovered from infection with virulent Wa human rotavirus (HRV) (mimic natural infection) had high numbers of intestinal IgA rotavirus-specific primary antibody-secreting cells (ASCs) and memory B-cells (to recall antigen) measured by ELISPOT assay, which correlated with complete protection against rotavirus challenge. Most short-term IgA memory B-cells were resident in the ileum, the major site of rotavirus replication. Spleen, not the bone marrow, was the major resident site for longer-term IgG memory B-cells. Candidate rotavirus vaccines evaluated in pigs for their ability to induce intestinal or systemic ASC and protection against rotavirus infection and diarrhea included attenuated live virus, inactivated virus, and baculovirus-expressed double-layered rotavirus-like particles (2/6-VLPs). In combination with those candidate vaccines, various adjuvants, delivery systems, and immunization routes were tested, including incomplete Freund's adjuvant for i.m. immunization, and a mutant Escherichia coli heat labile enterotoxin R192G (mLT) for i.n. immunization. It was shown that orally administered replicating vaccines were most effective for priming for intestinal IgA ASC and memory B-cell responses, but i.n. administered non-replicating 2/6-VLPs plus mLT were effective as booster vaccines. We conclude that protective immunity depends on the magnitude, location, viral protein-specificity, and isotype of the antibody responses induced by vaccination. Therefore highly effective enteric viral vaccines should: (i) induce sufficient levels of intestinal IgA antibodies; (ii) include viral antigens that induce neutralizing antibodies; and (iii) require the use of effective mucosal adjuvants or antigen delivery systems for non-replicating oral or i.n. vaccines.     

Studies on neonatal calf diarrhoea caused by rotavirus: transmission of the disease and attempted vaccination of colostrum-deprived calves

Mild to severe scouring could be produced in colostrum-deprived calves with tissue culture-adapted rotavirus and feacal material from field cases of calf diarrhoea. The feaces of experimentally infected calves contained rotavirus for at least 3 days. Pathogenic bacteria were presented in one calf only and this calf also showed the most severe gastroenteritis. Eight calves were vaccinated with a live rotaviral calf diarrhoea vaccine and subsequently challenged with infective rotavirus. Mild scouring was observed after vaccination, but the calves remained normal after challenge. Rotavirus particles were detectable in the faeces for a few days after vaccination and challenge.     

Epizootic diarrhoea of adult cattle associated with a coronavirus-like agent

An epizootic of acute diarrhoea of adult cattle occured in Japan during the winter of 1976 to 1977. A majority of adult cattle clinically diagnosed as having the disease, showed a significant rises in antibody titres to bovine coronavirus, whereas only a smal; minority showed serological evidence of recent infection with calf rotavirus, bovine adenovirus type 7, parainfluenza virus type 3, or bovine viral diarrhoea-mucosal disease virus. A coronavirus-like agent was detected by electron-microscopy in faecal material from a cow with diarrhoea, and was subsequently isolated in primary bovine kidney cell cultures.     

A new polyvalent vaccine against enteral infections in newborn calves

The most frequent microbial causative agents of massive diarrheas in new-born calves kept on large cattle farms in the CSSR are rotaviruses, coronaviruses and enterotoxigenic strains of E. coli, manifesting themselves as complicated virus-bacterial infections. An inactivated polyvalent adjuvant vaccine has been developed for the prevention and specific prophylaxis of these enteral infections; the vaccine contains bovine rotavirus, bovine coronavirus and three enterotoxigenic serotypes of E. coli with protective antigen K 99. The rotavirus and coronavirus are propagated on the stable cellular line MDBK and inactivated with 0.2% formalin, the Escherichia strains are submersed in the MINCA culture medium during their cultivation and inactivated with 0.5% formalin. The vaccine was prepared as a blend of the same amounts of rotavirus and coronavirus and of such an amount of bacterin so that 1 ml of the vaccine will contain 10(9) bacteria. One part of oil adjuvant was added to five parts of the virus-bacterial blend and the blend was homogenized in the Ultraturax apparatus. The vaccine is to be used for immunization of pregnant cows and heifers; in these animals it induces the production of specific antibodies to all antigens contained in the vaccine. Its immunogenic effects were checked in 32 calves and 38 cows in the herds with the occurrence of diarrheas caused by both enteropathogenic viruses and enterotoxigenic escherichia. It was demonstrated that the inactivation did not influence in either of the viruses the process of inducing the production of specific antibodies, and the antibody response of the calves and heifers after application of 2 ml of complete inactivated vaccine was equally strong as after application of live vaccine containing only rotavirus and coronavirus. The level of the rotavirus antibodies increased on the average 30 times and 200 times, coronavirus antibodies twice and four times. The antibody response to coronavirus was negatively influenced by the relatively high levels of antibodies before vaccination. The antibody response to antigen K 99 was expressive in all cases.     

Rotavirus infections in calves in dairy herds

Neonatal calf diarrhoea was studied in 115 calves of one dairy herd from January 1976 to June 1977. Two syndromes could be distinguished: a mild and short lasting 'early diarrhoea' within the first three days of life and a usually more severe 'late diarrhoea' from the fourth to the 14th day of life. The latter type of diarrhoea occurred almost exclusively during the first half of the year. Thirty-four out of 45 calves with late diarrhoea excreted rotaviruses, whereas only one of 34 calves with early diarrhoea excreted virus. In addition, rotavirus excretion was found in 11 calves that either remained healthy or had recovered from diarrhoea before virus was first detected. Similar findings were obtained in the spring of 1978 but in addition bovine coronavirus was recovered from four calves with late diarrhoea and from three healthy calves. One faecal sample obtained from a calf with diarrhoea on day 2 yielded K99+ Escherichia coli. Nearly all cows excreted rotavirus-specific antibodies in their colostrum but no relationship was found between the initial colostral antibody titre against rotavirus and the development of rotavirus-associated diarrhoea in the calf.     

Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus

Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.