First Report of Fusarium equiseti Causing Vascular Wilt of Cumin in India.

Cumin (Cuminum cyminum) is one of the important spices in the world after black pepper. Cumin is mainly used in flavoring foods for its distinctive aroma and ayurvedic medicines. The production of cumin seeds in India is estimated to be around 0.15 million tons annually. Wilt symptoms were observed on field-grown cumin during the winter of 2009 and 2010. Diseased plants exhibited symptoms including wilted leaves, stunted growth, and eventually, death. In severe cases, approximately 65% of the plants in the field died. Isolations of the pathogen were made from the discolored tissues on potato dextrose agar (PDA) and Rose Bengal after disinfestations in 0.1% HgCl2 for 2 min and dipping in 70% ethanol for 10 s. Petri dishes were then incubated in complete darkness at 26°C for 7 days. Typical growth characters observed were development of abundant white aerial mycelium that turned peach orange by incubating under light. Microconidia were single-celled, hyaline, non-septate and ovoid, and ranged from 9.5 to 12.5 × 3.5 to 5.25 µm. Macroconidia were mostly two- to three-septate, slightly curved at apex, and ranged from 28.0 to 30.5 × 3.5 to 5.25 µm. The fungus was identified as Fusarium equiseti based on colony characters and spore morphology (3). The identity was further confirmed by the Fungal Identification Service, Mycology and Plant Pathology Group Agharkar Research Institute, Pune, India (Accession No. NFCCI-2157). The ITS (internal transcribed spacer) region of rDNA was amplified by polymerase chain reaction (PCR) with primers ITS1/ITS2 and sequenced (2). BLASTn analysis of the sequence obtained showed a 99.78% homology with F. equiseti at NCBI and FUSARIUM-ID v. 1.0 (1). The sequence was deposited at GenBank (Accession No. JN014954). Pathogenicity tests were conducted on 10 healthy 1-month-old seedlings of cumin in a moist chamber. Plants were separately inoculated in pots with sterilized soil and 10 ml of F. equiseti isolate spore suspension (107 conidia/ml) at 25°C for 7 to 10 days. Control plants were inoculated with sterile soil without spore suspension. Within 10 days, inoculated plants developed leaf wilt, stunted growth, discolored vascular tissue on stems, and finally died, which is similar to that observed in the field. Control seedlings were symptom free. Koch's postulates were fulfilled by reisolating the fungal pathogen, which was identified as F. equiseti causing vascular wilt on cumin reported in Israel (4). However; F. oxysporum f. sp. Cumini is the first reported vascular wilt of cumin. To our knowledge and on the basis of the literature, this the first report of vascular wilt of cumin caused by F. equiseti in India. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) M. Korabecna. In: Communicating Current Research and Educational Topics and Trends in Applied Microbiology, p. 783, 2007. (3) J. F. Leslie and B. A. Summerrell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) R. Reuveni. Plant Dis. 66:498, 1982.

Sendai viral pneumonia in aged BALB/c mice.

Sendai virus (SV) infection in aged BALB/c mice was evaluated as a natural model for age-associated susceptibility to viral pneumonia. Young (2 month-old) and aged (22-24 month-old) BALB/c mice were inoculated intranasally with 100 median pneumonia doses (PD50) of SV and examined at 6, 10, and 20 days by virus titration, immunohistochemistry, histopathology, and serology. The aged mice had significantly higher virus titers in lung, prolonged infection, delayed development, and resolution of pneumonia and significantly lower serum antibody titers. In a second experiment, the responses of young mice were compared to intermediate-aged mice (11-13 and 17-18 months old). The intermediate-aged mice had some characteristics of young mice and others of aged mice. The results indicate that SV infection can be used to study aging-associated susceptibility to a pneumotropic virus in a natural host, and that susceptibility of mice to viral pneumonia increases gradually during aging.

Infrequent shedding and transmission of herpesvirus simiae from seropositive macaques.

The epizootiologic properties of Herpesvirus simiae (B virus) were studied in singly housed macaques (Macaca mulatta and M. fascicularis) in a biomedical vivarium to determine whether commonly encountered environments and procedures such as quarantine, breeding, Caesarean section, parturition, and social stress induced virus shedding and transmission. Macaques were tested serologically and for infectious virus. Oral, conjunctival, and vaginal swab samples were obtained repeatedly. Virus excretion was not detected during a 7-week quarantine of 32 newly acquired, singly housed animals tested every other week for 6 weeks, and none of 19 seronegative animals from this group seroconverted during 7 weeks in quarantine. No virus shedding was detected in 16 seropositive animals tested weekly for 3 weeks after Caesarean section or normal parturition or in 11 seropositive animals following introduction of new males to animals rooms. One animal seroconverted after repeated breeding of seropositive animals to seronegative partners. Fifty-three singly housed offspring remained seronegative for up to 10 years, even if born to seropositive dams, and only 1 of 86 singly housed animals less than 7 years old was seropositive. These results suggest that shedding of B virus from seropositive macaques is uncommon, when subjected to common laboratory procedures or environments, and that transmission is rare in singly housed animals. These results may be useful in establishing B virus-free colonies of macaques.

Infectivity, disease patterns, and serologic profiles of reovirus serotypes 1, 2, and 3 in infant and weanling mice.

The oronasal median infectious doses of reovirus serotypes 1, 2, and 3 were established in infant and weanling Sencar mice on the basis of disease expression and seroconversion. Infant mice were susceptible to infection with low doses of all three serotypes, whereas weanling mice were comparatively resistant to infection. Uniform transmission of virus to cagemates or mothers of infants did not occur, indicating low contagiousness of all three virus serotypes. The comparative susceptibility of 2-day-old Sencar mice to disease was examined following oronasal inoculation with reovirus 1, 2, or 3. Tissues were collected on days 3, 5, 7, 9, 14, 16, and 21 after inoculation for virus isolation, histologic examination, and serologic analysis. Disease patterns in infant mice were distinctly different among reovirus serotypes. Reovirus 3 induced severe disease, with focal myocarditis, hepatitis, diffuse encephalitis, and generalized lymphoid depletion, whereas reovirus 1 induced a similar pattern, but much milder disease. In contrast, reovirus 2 induced mild transient enteritis without lesions in other organs. Sera from experimentally infected mice were tested in virus serotype-specific enzyme immunoassays. Cross reactivity of antibody among the three virus serotypes was found, but antibody titers were always highest with the homologous antigen. These studies confirm that infant laboratory mice are susceptible to infection with all three serotypes of virus; weanling mice are comparatively resistant to infection and disease; the viruses induce different patterns of disease in infant mice; and infecting virus serotypes can be distinguished serologically by enzyme immunoassay.

In vivo studies with an "orphan" parvovirus of mice.

A virus antigenically related to, but distinct from, minute virus of mice was assessed for infectivity in neonatal and weanling random-bred mice and was equally infectious for both age groups. The virus, designated a mouse "orphan" parvovirus (OPV), was also localized in tissues of experimentally infected random-bred, inbred, and immunodeficient mice by in situ hybridization. Hybridization signal was seen in exocrine and endocrine pancreas, abdominal lymph nodes, mesentery, intestine, and sporadically in other tissues of Sencar, C3H, and DBA mice inoculated as infants. In adult BALB/c severe combined immunodeficient (scid) mice, signal was seen in lung, liver, spleen, lymph nodes, and intestine but not in pancreas. Transmission of OPV by Sencar mice inoculated as infants was intermittent, whereas transmission by Sencar mice inoculated as weanlings was consistent during the first 2 weeks both by direct contact and by exposure to soiled bedding. The longest duration of transmission was 6 weeks among mice inoculated as infants. The results implicate a role for urinary, fecal, and perhaps respiratory excretion of virus, depending on host genotype and route of virus exposure. They also suggest that evaluation of pancreatic and immune function during acute infection is warranted.

Ectromelia virus replication in major target organs of innately resistant and susceptible mice after intravenous infection.

The kinetics of ectromelia virus replication in the spleen and liver and of alpha/beta interferon production in the spleen were determined during the first 3 days after intravenous infection with the virulent Moscow strain in resistant C57 BL/6 and susceptible DBA/2 mice. Virus replication in the spleen as measured by assays for virus DNA and infectious centers was suppressed in C57BL/6 mice relative to DBA/2 mice within the first 1 or 2 days of infection. Infectious centers increased in DBA/2 mice but not in C57 BL/6 mice. Differences in virus replication between strains were less discrete when spleens were assayed for infectious virus than when they were assayed for infectious centers because infectious centers of most C57 BL/6 mice had more infectious virus than infectious centers of DBA/2 mice. Virus replication in the liver, the major target organ, as measured by virus DNA and infectious virus assays, was suppressed in C57 BL/6 mice relative to DBA/2 mice 3 days after infection but not before that interval. The results indicate that genetic control of ectromelia virus replication begins within the first 1 or 2 days of infection in the spleen but is delayed in the liver and that genetic control is directed at the prevention of virus spread more than at virus replication.

Serial backcross analysis of genetic resistance to mousepox, using marker loci for Rmp-2 and Rmp-3.

At least three genes from C57BL/6 mice that mediate dominant resistance to lethal mousepox were isolated and transferred onto a susceptible DBA/2 background. Three [(C57BL/6 x DBA/2)F1 x DBA/2] male mice that survived infection were selected as founders on the basis of different complements of marker loci for two resistance genes, Rmp-2r (Hc1) and Rmp-3r (H-2Db). They were crossed with DBA/2 mice, male progeny were infected with ectromelia virus, and the cycle was repeated with surviving male progeny through seven backcross generations. Two founders carried a marker locus for Rmp-2r or Rmp-3r, and the third carried neither marker locus. Resistance pedigrees were analyzed for passage of marker loci. From the three founders, resistance was passaged through multiple generations, producing backcross lines with intermediate-male-resistance phenotypes (20% resistant). Females of backcross lines with intermediate male resistance had high resistance (> 50%). High-resistance backcross lines (40% male resistance) also developed from the founders that carried marker loci for Rmp-2r and Rmp-3r, and marker loci were passaged through all generations of high resistance but not intermediate-resistance lines. About one-third of all resistant mice in high-resistance lines sired by mice that carried marker loci for Rmp-2r and Rmp-3r did not carry the respective marker locus. In lines that carried Rmp-2r, this was apparently not the result of recombination between Rmp-2r and Hc1, because Rmp-2 was not in the predicted location on chromosome 2 and because mice that did not inherit Hc1 transmitted significantly less male resistance than Hc1-positive mice, although female resistance remained high. These results confirmed that C57BL/6 mice have redundant resistance mechanisms, two of which are controlled at least in part by Rmp-2r and Rmp-3r, and provided evidence for a fourth resistance gene, herein presumptively named Rmp-4, which protects females more than males and which may be epistatic to Rmp-2.

Chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality.

Four genetic loci were tested for linkage with loci that control genetic resistance to lethal ectromelia virus infection in mice. Three of the loci were selected because of concordance with genotypes assigned to recombinant inbred (RI) strains of mice derived from resistant C57BL/6 and susceptible DBA/2 (BXD) mice on the basis of their responses to challenge infection. Thirty-six of 167 male (C57BL/6 x DBA/2)F1 x DBA/2 backcross (BC) mice died (22%), of which 27 (75%) were homozygous for DBA/2 alleles at Hc and H-2D. Twenty-eight percent of sham-castrated and 6% of sham-ovariectomized BC mice were susceptible to lethal mousepox, whereas 50% of gonadectomized mice were susceptible. There was no linkage evident between Hc or H-2D and loci that controlled resistance to lethal ectromelia virus infection in 44 castrated BC mice. Mortality among female mice of BXD RI strains with susceptible or intermediate male phenotypes was strongly correlated (r = 0.834) with male mortality. Gonadectomized C57BL/6 mice were as resistant as intact mice to lethal ectromelia virus infection. These results indicate that two gonad-dependent genes on chromosomes 2 and 17 and one gonad-independent gene control resistance to mousepox virus infection, that males and females share gonad-dependent genes, and that the gonad-independent gene is fully protective.

Duration of protection from reinfection following exposure to sialodacryoadenitis virus in Wistar rats.

Wistar rats [Cr1:(WI)BR] were inoculated intranasally with approximately 10(3) median mouse lethal infective doses of sialodacryoadenitis (SDA) virus. Animals were subsequently selected at random, removed to a separate isolation room, and reinfected with SDA virus at 3, 6, 9, 12 or 15 months. Pre- and postinoculation serum samples were collected from all animals during the course of the study and evaluated for antibody titers to SDA virus. All experimental, control and sentinel animals, following inoculation with SDA virus, were necropsied and examined for lesions consistent with SDA. Salivary gland lesions were minimal to absent in rats reinfected with SDA virus for up to 12 to 15 months after the initial exposure and minimal to moderate in the respiratory tract at 12 or 15 months. SDA-associated lesions were extensive in age matched control animals examined at each time period of reinfection with SDA virus. Thus, prior exposure to SDA virus did protect against the development of typical salivary gland lesions for up to 15 months. Recovered animals were evaluated for their ability to transmit the virus following reinfection. Rats reinfected at 6 or 9 months were infectious to their naive cage mates. The results indicate that reinfection with homologous rat coronavirus can occur as early as 6 months after the initial infection, and such rats can transmit the infection to contact controls.

Solasodine levels in Solanum sisymbriifolium Lam.

Solasodine content in berries, leaves and stem of S. sisymbriifolium, using three different procedures, was 0.93, 0.73 and 0.23% respectively. Further in chromatogram not only solasodine but three more alkaloids (Rf 0.22, 0.48 and 0.88) were found which could not be identified due to lack of authentic samples.

Evidence that NK cells and interferon are required for genetic resistance to lethal infection with ectromelia virus.

C 57 BL/6 mice developed resistance to lethal intravenous challenge with virulent (Moscow strain) ectromelia virus between 2 and 3 weeks of age. The fraction of C57 BL/6 mice in which virus was detected in spleen was significantly lower than for DBA/2 mice by day 3. Thereafter, C 57 BL/6 mice had significantly reduced virus titers in spleen compared with those of DBA/2 mice. Resistance was abrogated by treatment with anti-asialo GM1 gammaglobulin, which blocks NK cell activity, or with anti-interferon (IFN) alpha, beta. C 57 BL/6 mice carrying the bg/bg mutation, associated with a deficiency of NK cells, were highly susceptible to lethal infection as were athymic mice derived from a resistant genetic background. Virus titers in spleens of C 57 BL/6 mice treated with anti-asialo GM1 or anti-IFN alpha, beta were significantly higher 4 days after virus challenge than were titers in C 57 BL/6 mice treated with normal rabbit serum. The results strongly suggest that genetic resistance to lethal ectromelia virus infection requires non-specific host defenses such as NK cells and IFN alpha, beta that are activated during the first 3 to 4 days of infection.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. V. Genetics of resistance to the Moscow strain.

The genetics of resistance to the Moscow strain of ectromelia virus was examined in crosses derived from resistant C57BL/6 (B6) and susceptible DBA/2 (D2) mice. Infection with 10(1) to 10(5) PFU of virus resulted in mortalities of 90 to 100% of D2, 0% of B6 and 0 to 3% of (B6 x D2) F1 mice by day 21. Among F1 x D2 backcross progeny, 49% of male and 18% of female mice died. Reciprocal backcrossing did not alter male or female mortality rates. These data are consistent with a single autosomal dominant gene controlling resistance to ectromelia in male mice and at least one additional dominant sex-limited gene controlling resistance in female mice. Fewer male F2 mice died than were predicted based on single-locus control and 32% of recombinant inbred (RI) strains derived from B6 and D2 progenitors expressed non-parental phenotypes. Therefore, additional resistance genes, not expressed in backcross mice, were apparently expressed in F2 mice and RI strains.

Transmission of experimentally-induced rat virus infection.

The duration of transmission of rat virus (RV) infection was determined using Sprague-Dawley rats inoculated oronasally as juveniles (4 weeks old) or as infants (2 days old). Contact transmission from rats inoculated as juveniles was detected for 3 weeks, whereas transmission from rats inoculated as infants occurred for 10 weeks. Transmission continued for at least 7 weeks after seroconversion occurred in rats inoculated as infants. Two of three rats that had ceased to transmit infection harbored infectious virus as detected by explantation of kidney. Intrauterine transmission occurred only after pregnant dams were inoculated with large doses of virus and was more efficient when virus was inoculated intravenously than by the oronasal route. Enzyme immunoassay antibody titers to RV in offspring of previously infected dams decreased steadily during the first 13 weeks of life and 27 of 29 offspring tested by immunofluorescence assay at 12 or 13 weeks of age were seronegative. These results indicate that RV was transmitted by rats inoculated as infants for long periods after seroconversion occurred. They also suggest that the offspring of previously-infected dams were not infected. In utero transmission of RV-Y is unlikely to occur after oronasal inoculation unless rats are exposed to large doses of virus.

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. IV. Studies with the Moscow strain.

The pathogenesis and transmission of infection with the Moscow strain of ectromelia virus were studied in inbred mice. BALB/cAnNcr had high morbidity and mortality and C57BL/6Ncr (B6) mice had high morbidity and low mortality. Virus was detected in B6 mice for 2 weeks after subcutaneous (s.c.) inoculation and infected mice developed lesions compatible with acute mousepox. B6 inoculated by footpad transmitted infection to cagemates for up to five weeks and soiled cages that had housed infected mice were infectious for three weeks. S.c.-inoculated B6 mice also transmitted by contact for 2 weeks. Transmission was attributed to oronasal excretion of virus. Airborne transmission of infection between adjacent cages occurred at a low rate. Ectromelia virus-free progeny were derived from previously infected dams. These studies indicate that the highly virulent and infectious Moscow strain of ectromelia virus caused self-limiting infection in inbred mice and that direct contact is the most efficient means of transmission. These findings support the concept that mousepox can be contained by husbandry practices that minimize or eliminate the spread of infection by direct contact or fomites.

Effect of vaccination on the clinical response, pathogenesis and transmission of mousepox.

The effects of vaccination with the IHD-T strain of vaccinia virus on the course and severity of ectromelia virus infection was investigated in BALB/c mice. Protection from lethal mousepox occurred when mice were vaccinated and challenged on the same day and protection persisted for at least 9 months. Vaccinated mice were not protected from infection or from lesions, but necrotic lesions in vaccinated mice were usually mild and were accompanied by inflammation, whereas necrosis in unvaccinated mice was severe and not accompanied by inflammation. Inoculated feet of previously vaccinated mice contained infectious ectromelia virus for at least 28 days. Vaccinated-challenged mice transmitted infection to non-immune cagemates for up to 2 weeks, but only rarely transmitted virus to vaccinated cagemates. These results emphasize that vaccination protects mice against lethal mousepox, but it does not prevent infection. In addition, vaccination reduces, but does not eliminate, transmission of infection to non-immune and immune mice.

Elimination of mouse hepatitis virus from a breeding colony by temporary cessation of breeding.

During quarantine in a Trexler-type isolator, high mortality was noted among offspring in a breeding colony of C.B.-17 scid/+ mice. Histology and immunohistochemistry on tissues of surviving weanlings confirmed mouse hepatitis virus infection (MHV). Since MHV infection is reported to be acute and self limiting, elimination of infection was attempted by cessation of breeding for a 15-week period. F1 mice born thereafter were seropositive for MHV at 3 to 4 weeks old and seronegative 4 weeks later, attributed to decay of maternally-derived antibodies. F2 mice were seronegative for MHV at 3 to 9 weeks old. No deaths occurred in any litters. These results suggest that MHV can be eliminated from a colony by temporary cessation of breeding, as evidenced by seronegative progeny through the F2 generation.

Further evidence for papovavirus as the probable etiology of transmissible lymphoma of Syrian hamsters.

The role of hamster papovavirus as the etiology of transmissible lymphoma was investigated under strict conditions that prevented natural exposure to the lymphoma agent. In an initial experiment, 19 hamsters that were exposed naturally to transmissible lymphoma were placed in direct and indirect contact with weanling hamsters from an uninfected source. Lymphoma developed in the original infected hamsters as well as hamsters maintained in direct and indirect contact. In addition, one of the contact hamsters developed cutaneous epitheliomas, containing hamster papovavirus. Epithelioma homogenate was inoculated into primary hamster embryo cultures, in which hamster papovavirus replicated. Second and third passage tissue culture fluid containing hamster papovavirus induced lymphomas in suckling and weanling hamsters. Cell culture fluid from uninoculated embryo cultures was not oncogenic.

Stability of ectromelia virus strain NIH-79 under various laboratory conditions.

Ectromelia virus strain NIH-79 was suspended in fetal bovine serum (FBS), minimum essential medium, Hanks' base plus 10% FBS (MEMH + FBS), phosphate-buffered saline (PBS) or PBS plus 50% glycerol (PBS + G). Suspensions were held as liquids or as dry spots at various temperatures. Virus was most stable in FBS and least stable in PBS + G at 4 degrees C, room temperature (23-25 degrees C) or 37 degrees C. Virus held at 4 degrees C was more stable than virus held at higher temperatures, irrespective of supporting medium. Dried spots of blood or serum from ectromelia virus-infected mice remained infectious at room temperature for 11 days and 4 days, respectively. Dried spots of FBS that contained virus were infectious for 5 days, whereas virus retained infectivity for 1 day after drying in other media. Virus was inactivated completely in 10% serum in PBS exposed to 60 degrees C for 30 minutes. Virus was inactivated completely in slices of infected liver and spleen immersed in 10% neutral buffered formalin for 20 hours. These results show that the stability of ectromelia virus strain NIH-79 is medium and temperature dependent and that rapid inactivation occurs after treatments routinely used in diagnostic and research procedures.