Aerosol transmission of an avian influenza H9N2 virus with a tropism for the respiratory tract of chickens.

A low pathogenic avian influenza virus (LPAI H9N2) was administered to 3-wk-old chickens by aerosol exposure, intranasal inoculation, and by oral inoculation. Tests for virus were by in ovo assay and by real-time reverse-transcriptase PCR. The aerosol dosage was determined by aerosolizing virus into a chamber when it was empty and when it contained chickens. Air was collected and the amount of virus inhaled was estimated to be about 18% of the total body uptake. In transmission studies, tests for virus were conducted on oropharyngeal or cloacal swabs. The 50% infectious dose (ID50) for aerosolized virus was about 2 log 10 and 5 log 10 lower than by nasal or oral inoculation, respectively. The recovery rate was higher from swabs of the oropharyngeal region than from the cloacal region (P < 0.05). For horizontal transmission studies, uninfected chickens were held in isolators with seeders that had been inoculated intranasally with the H9N2 virus. Chickens exposed by indirect contact were separated by screens from the seeders. In another isolator those directly exposed were intermingled with the seeders. During the 10-day test period, none of the chickens developed symptoms of disease, but infection was detected as early as 4 and 7 days in the indirectly and directly exposed groups, respectively. These findings suggested that aerosol transmission of viruses similar to LPAI H9N2 could efficiently occur, at least over short distances.

The fate of recombinant plasmids during composting of organic wastes.

Composting was investigated as a means for safe disposal of organic waste containing bacteria that carry transgenes in recombinant plasmids. To generate model recombinant plasmids, a mobile IncQ plasmid, RSF1010, and a non-mobile plasmid, pGFP, were genetically modified to carry a DNA segment encoding both green fluorescent protein and kanamycin resistance and were designated as RSF1010-GFPK and pGFPK. Escherichia coli (E. coli) C600 harboring these plasmids were inoculated into chicken manure specimens that were placed in compost at 20 and 60 cm from the bottom of a 1.0-m high compost bin. Control specimens were held at ambient temperature. By day 10, compost temperatures at the lower and upper levels of the bin had reached 45.3 and 61.5 degrees C, respectively, and at both levels the target E. coli had been inactivated and the plasmids had lost their capacity to be transformed or mobilized. Furthermore, based on real time Polymerase chain reaction (PCR), the transgene fragments along with the host chromosomal DNA fragment from specimens at the upper level had been degraded beyond the detection limit. However, at the lower level where temperatures remained below 48 degrees C these fragment persisted to day 21. At ambient temperatures (0-8 degrees C), the E. coli, plasmids and the transgene fragments persisted in manure specimens throughout the 21 day test period. The study showed the potential for composting as a safe procedure for disposal of bacteria carrying transgenes in recombinant plasmids.

Evidence for degradation of abnormal prion protein in tissues from sheep with scrapie during composting.

This study investigated whether the abnormal prion protein (PrP(Sc)) in tissues from sheep with scrapie would be destroyed by composting. Tissues from sheep naturally infected with scrapie were placed within fiberglass mesh bags and buried in compost piles for 108 d in experiment 1 or 148 d in experiment 2. The temperature in the compost piles rose quickly; it was above 60 degrees C for about 2 wk and then slowly declined to the ambient temperature. Before composting, PrPSc was detected in all the tissues by Western blotting. In experiment 1, PrPsc was not detected after composting in the tissue remnants or the surrounding sawdust. In experiment 2, 1 of 5 specimens tested negative after composting, whereas PrP(Sc) was detected in the other 4 bags, though in reduced amounts compared with those before composting. Tissue weights were reduced during composting. Analysis of the tissue remnants for microbial 16S ribosomal DNA demonstrated that there were more diverse microbes involved in experiment 1 than in experiment 2 and that the guanine and cytosine content of the microbial 16S DNA was higher in the specimens of experiment 1 than in those of experiment 2, which suggests greater dominance of thermophilic microbes in experiment 1. These results indicate that composting may have value as a means for degrading PrP(Sc) in carcasses and other wastes.

Relationship between Marek's disease and the time course of viral genome proliferation in feather tips.

A total of 114 male chickens from three sire families of a commercial cross of White Leghorn chickens were infected with RB-1B Marek's disease (MD) virus at 21 days of age by exposing them to chickens previously inoculated with MD virus. The presence of virus in feather tips, feather pulp, and MD viral antibodies indicated all chickens became infected. The first virus-positive chickens were observed at 12 days postexposure (dpe). The frequency reached a maximum at 27 dpe and then decreased. At 80 dpe, when the experiment was terminated, no viral DNA was detected in the feather pulp of the surviving chickens (82%). Death from MD was first observed at 38 dpe and reached 18% by the end of the experiment, with spleen lesions being the major MD lesion. The viral genome titers in spleen extracts of chickens with MD lesions was negatively correlated with the time of death, and, similar to feather pulp, none of the surviving chickens was virus positive at the end of the experiment. Quantization of the viral genome titers in feather tip extracts at 27 and 38 dpe revealed a positive correlation with the presence of MD lesions, but only in the declining phase (38 dpe) and not at the peak (27 dpe) of the viral titer. Sire effects were significant, indicating the presence of genetic factors that affect viral proliferation. Again, significance was only observed at 38 dpe and not at 27 dpe. The results indicate that, in this commercial line, 1) all chickens were susceptible to infection via contact exposure, 2) all surviving chickens recovered from the viral infection, and 3) it is not sufficient to measure viral titers at a single time point when using viral titers as an endpoint for MD susceptibility.

Development of a real-time PCR assay using SYBR Green chemistry for monitoring Marek's disease virus genome load in feather tips.

Feather follicles of birds infected with Marek's disease virus (MDV) serve as the sole source of infectious virus particles. The present study was aimed at developing a SYBR Green real-time PCR assay to detect and quantify MDV loads in feather tips targeting meq gene of the virus. The assay had a dynamic range of 8 logs, mean inter- and intra-assay coefficient variation (CV) of <5% and minimum detection limit of 15 MDV genome copies when plasmid DNA was used as the template. The sensitivity of the assay was compared with that of the conventional PCR technique and found to be 2.5-10 times more sensitive than the conventional PCR technique. The assay was validated using feather tip DNA preparations derived from chickens infected with 250 plaque forming units (PFU) of RB1B strain of MDV and sampled on days 7, 14, 21 and 28 post-infection (p.i.) along with uninfected chickens. MDV genome was quantifiable in feather tips of infected birds by day 7 p.i. and the number of MDV copies peaked by day 14 p.i., but then gradually decreased by day 28. This reliable real-time PCR assay may be used for monitoring MDV genome loads in tissues of experimentally or naturally infected birds.

The fate of the recombinant DNA in corn during composting.

In order to make regulations that safeguard food and the environment, an understanding of the fate oftransgenes from genetically modified (GM) plants is of crucial importance. A compost experiment including mature transgenic corn plants and seeds of event Bt 176 (Zea mays L.) was conducted to trace the fate of the transgene cryIA(b) during the period of composting. In bin 1, shredded corn plants including seeds were composted above a layer of cow manure and samples from the corn layer were collected at intervals during a 12-month period. The samples were tested for the transgene persistence and microbial counts and also the compost was monitored for temperature. In bin 2, piles of corn seeds, surrounded by sheep manure and straw, were composted for 12 months. A method combining nested polymerase chain reaction (PCR) and southern hybridization was developed for detection of the transgene in compost. The detection sensitivity was 200 copies of the transgene per gram of dry composted corn material. Composting commenced on day 0, and the transgene was detected in specimens from bin 1 on days 0 and 7 but not on day 14 or thereafter. The transgene in corn seeds was not detectable after 12 months of composting in bin 2. Temperatures in both bins rose to about 50 degrees C within 2 weeks and remained above that temperature for about 3 months, even when the ambient temperature dropped below -20 degrees C. Extracts from compost were inoculated onto culture plates and then were incubated at 23 to 55 degrees C. Within the first 2 weeks of composting in bin 1, the counts of bacteria incubated at 55 degrees C increased from 3.5 to 7.5 log10, whereas those incubated at 23 degrees C remained at about 7.5 log10. The counts of fungi incubated at 45 degrees C increased slightly from 2.5 to 3.1 log10, but those incubated at 23 degrees C decreased from 6.3 to 3.0 log10. The rapid degradation of the transgene during composting of Bt corn plants suggested that the composting process could be used for safe disposal of transgenic plant wastes.

Public health implications related to spread of pathogens in manure from livestock and poultry operations.

During the 20th century, food animal agriculture grew from small operations, where livestock (cattle, sheep, and swine) and poultry (chickens and turkeys) had access to free range, to large operations where animals and poultry were concentrated and confined to feed lots or buildings. The quantity of manure produced by confinement animals in the United States has been estimated to be in excess of 61 million tons of dry matter per year, and another report states that 1.2 billion tons of manure are produced by cattle annually in the United States (US Senate Agricultural Committee, 1998). As urban developments have come closer to livestock operations, there has been increasing public concern for the impact of the latter on public health and the environment. Although management practices for livestock production have increased in efficiency, insufficient attention has been given to managing and utilizing wastes so that they benefit rather than pollute the environment. Animal manure includes urine and various bodily secretions such as those from the nose, vagina, and mammary glands. Dust from animals and manure may be blown from buildings by powerful fans, and manure is often piled near the animal quarters or is spread on land in solid or liquid form. Public concerns associated with disposal of animal manure include objectionable odors, flies, excessive levels of phosphorous and nitrogen, and the potential for spread of human pathogens. It has been observed that despite linkages between outbreaks of gastroenteritis in humans and livestock operations, the importance of animal manure in the spread of infectious agents tends to be underestimated.

Evidence for virus closely related to avian myeloblastosis-associated virus type 1 in a commercial stock of chickens.

A two-round nested polymerase chain reaction assay detected Rous associated virus-1 (RAV-1), a prototype laboratory strain of avian leukosis virus of subgroup A (ALV-A). Surprisingly, the test failed to detect three field isolates of ALV-A but did detect virus in one commercial stock of chickens (stock F). The sequence analysis of a core of 290 nucleotides of the env gene gave evidence that the virus from stock F was closely related to avian myeloblastosis-associated virus type one (MAV-1). Other primers were used to amplify and sequence a 1491 nucleotide fragment of the env gene, and a 1245 nucleotide portion of this sequence used for a phylogenetic comparison. These analyses on 10 chickens gave evidence that six were infected with MAV-1-like virus and three with RAV-2 (subgroup B virus), and one chicken with a mixture of the two viruses. Tests with primers designed specifically for the MAV-1 sequence at a pre-determined target site and a second primer designed specifically for RAV-2 at the same site gave further evidence that the viruses isolated from stock F were closely related to one or other of these two viruses.