Sukhbir K. Grewal

Persistence of Mycobacterium avium subsp. paratuberculosis and Other Zoonotic Pathogens during Simulated Composting, Manure Packing, and Liquid Storage of Dairy Manure

ABSTRACT Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55°C, manure packing at 25°C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 106 CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO2, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55°C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.

Differences in susceptibility of introduced and native white grub species to entomopathogenic nematodes from various geographic localities

Abstract Invasive, non-native, white grubs (Coleoptera: Scarabaeidae) cause significant damage in urban landscapes. Although the lack of natural enemies in their new home is often suggested as an important factor in the establishment and spread of invasive species, the potential of incumbent generalist parasites and pathogens to delay their establishment and spread has not been explored. We compared the susceptibility of the introduced Popillia japonica and the native Cyclocephala borealis to 16 species and strains of entomopathogenic nematodes isolated from within or outside the geographic ranges of the two scarabs. We found large variation in the virulence of the species/strains of nematodes with over 50% mortality of P. japonica produced by Heterorhabditis zealandica strain X1 and H. bacteriophora strain GPS11 and of C. borealis by H. zealandica and H. bacteriophora strains KMD10 and NC1. Heterorhabditis indica and H. marelatus caused less than 20% mortality of both scarab species. When considered as a group the nematode species and strains from within and outside the geographic ranges of either P. japonica or C. borealis did not differ in virulence towards either scarab species. Dose response studies with selected nematode species and strains against P. japonica and two additional non-native species Anomala (Exomala) orientalis and Rhizotrogus majalis and the native C. borealis indicated that R. majalis was the least susceptible and P. japonica and A. orientalis were as susceptible as the native C. borealis. Heterorhabditis zealandica was significantly more virulent than any other species or strain against P. japonica with a LC50 of 272 IJs/grub. The LC30 and LC50 values for H. zealandica were also the lowest among the four nematode species/strains tested against A. orientalis and C. borealis. The LC50 values for H. zealandica and H. megidis (UK strain) were significantly lower for the native C. borealis than the introduced A. orientalis. H. zealandica also showed the highest penetration efficiency and the lowest encapsulation in P. japonica and C. borealis grubs. Results suggest that the introduction of the exotic H. zealandica into the front-line states with respect to the movement of P. japonica and A. orientalis should be explored as a tactic to delay their establishment and spread. The results also suggest that the manipulation of the indigenous H. bacteriophora populations may help in delaying spread and mitigating losses caused by the invasive grub species.

Susceptibility of North American Native and Non-native Slugs (Mollusca: Gastropoda) to Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Phasmarhabditis hermaphrodita caused significant mortality of the two native species, Deroceras laeve and Leidyula floridana, and of one introduced species D. reticulatum, but not of the other three introduced species, Arion hortensis, A. subfuscus and Limax maximus. Even the juvenile stages of A. subfuscus and L. maximus showed no mortality in nematode treatments. However, treatments with nematodes resulted in rapid and strong feeding inhibition in all six species. Surviving slugs resumed feeding when fresh food was provided. This study expands the host range of P. hermaphrodita to include a new family Vaginulidae and demonstrates the inability of P. hermaphrodita to cause mortality of A. hortensis, A. subfuscus and L. maximus. These three species use feeding suspension as an evasive behavior to escape nematode infection.

Changes in diversity of cultured bacteria resistant to erythromycin and tetracycline in swine manure during simulated composting and lagoon storage.

This study investigated the impact of composting and lagoon storage on survival and change in diversity of tetracycline‐resistant (Tcr) and erythromycin‐resistant (Emr) bacteria and the resistance genes they carry in swine manure. Treatments were arranged as a 2 × 2 factorial design: composting vs lagoon storage and 0 vs 1% Surround WP Crop Protectant (a clay product) in three replicates. After 48 days of treatments, resistant bacteria were enumerated by selective plating and identified by 16S rRNA gene sequencing. The erm and the tet gene(s) carried by the resistant isolates were screened using class‐specific PCR assays. The plate counts of Tcr and Emr bacteria decreased by 4–7 logs by composting, but only by 1–2 logs by the lagoon treatment. During the treatments, Acinetobacter gave way to Pseudomonas and Providencia as the largest resistant genera. The clay product had little effect on survival or diversity of resistant bacteria. Of six classes of erm and seven classes of tet genes tested, changes in prevalence were also noted. The results indicate that composting can dramatically shift Tcr and Emr bacterial populations, and composting can be an effective and practical approach to decrease dissemination of antibiotic resistance from swine farms to the environment.

Non-Susceptibility of Earthworm Eisenia fetida to the Rhabditid Nematode Phasmarhabditis hermaphrodita, a Biocontrol Agent of Slugs

The rhabditid nematode Phasmarhabditis hermaphrodita is a lethal parasite for slugs that is sold commercially in Europe under the trade name Nemaslug™. We evaluated the effects of P. hermaphrodita on the earthworm Eisenia fetida (Savigny). Adults of E. fetida were exposed in one-liter glass beakers to P. hermaphrodita at three concentrations (1×, 10× and 50× of the field recommended rate of 3×109 billion nematodes/ha) during a 14-day period in an artificial soil substrate. The average body weight, burrowing behavior, mortality and other clinical signals of the earthworms were recorded at 0, 7, and 14 days after exposure to the treatments. In addition, injured earthworms (posterior ends removed) were exposed to the 10× field recommended rate of the commercial formulation. Neither intact nor injured E. fetida showed susceptibility to the slug-parasitic nematode P. hermaphrodita during the 14 days of exposure even at concentrations 10 and 50 times higher than the label dose. However, the worms in the attenuated control (autoclaved formulation) had higher mortality and lost less weight compared to the other treatments. Under the conditions of the test, we conclude that the use of the commercially available strain of P. hermaphrodita is safe to E. fetida.

Effect of osmotic desiccation on longevity and temperature tolerance of Phasmarhabditis hermaphrodita (Nematoda: Rhabditidae)

Limited storage stability severely restricts the biological control potential of slug-parasitic nematodes. In a series of experiments, we evaluated the effects of temperature and osmotic desiccation on the short- and long-term survival of the slug-parasitic nematode Phasmarhabditis hermaphrodita. Nematode survival in petri dishes at 1,500 infective juveniles/ml did not differ significantly at 5, 10, and 15 C but declined rapidly at 25 and 30 C. At 25 C about 50% of the nematodes survived for 4 wk, but at 30 C no nematode survived past 1 day. About 50% of the nematodes survived for 32 wk at 20 C. About 35–40% of the nematodes survived up to a year at 5, 10, and 15 C. Phasmarhabditis hermaphrodita showed poor survival under osmotic desiccation in glycerol with 15 and 20% glycerol significantly reducing survival at 5 and 15 C. Although the nematodes tolerated 10% glycerol, this level of desiccation also did not enhance long-term survival at either 5 or 15 C. There was a significant decrease in nematode survival in 10% glycerol at 25 C during the first 2–3 wk, but about 16% of the nematodes survived for 6 wk in 10% glycerol as compared with only 1% survival in water. The greatest benefit of osmotic desiccation in glycerol was observed in the enhanced survival of P. hermaphrodita at temperature extremes. Over 96% of the nematodes survived a 6-hr exposure to 35 C in 10% glycerol, whereas only 9% survived in water. Similarly, over 90% of the nematodes survived an exposure to −20 C for 4 hr in 10% glycerol, but less then 2% survived in water. We conclude that 5–15 C is an optimum temperature range for the storage of P. hermaphrodita. We also conclude that osmotic desiccation in 10% glycerol can substantially increase survival of P. hermaphrodita at temperature extremes (35 and −20 C) for short periods but has no effect on nematode longevity at the optimum temperature range of 5–15 C.