A survey for Cyclospora spp. in Kenyan primates, with some notes on its biology.

From March 1999 through August 2000, 511 stool samples collected from 11 different primate species in 10 geographically distinct locations in Kenya, East Africa, were screened for the presence of Cyclospora spp. oocysts. Positive samples (43/102, 42%) were identified in vervet monkeys (Cercopithecus aethiops) in 4 of 4 locations; 19/206 (9%) in yellow and olive baboons (Papio cynocephalus, P. anubis, respectively) in 5 of 5 locations; and 19/76 (25%) in black and white colobus monkeys (Colobus angolensis, C. guereza, respectively) from 2 of 3 locations. DNA sequences obtained from 18 S rRNA coding regions from respective subsets of these positive samples were typed as Cyclospora cercopitheci (samples from Cercopithecus aethiops). Cyclospora papionis (samples from Papio cynocephalus and P. anubis), and Cyclospora colobi (samples from Colobus angolensis and C. guereza). Cyclospora oocysts were not detected in samples collected from patas, highland sykes, lowland sykes, blue sykes, DeBrazza, or red-tailed monkeys. A coded map showing the geographic location of the collected samples is given. Stool samples from 1 troop of vervet monkeys were collected over a 12-mo period. Positive samples ranged between 21 and 63%. These results suggest that there is no strongly marked seasonality evident in Cyclospora infection in monkeys as has been noted in human infection. This is further confirmed by the recovery of positive samples collected from vervet monkeys, baboons, and colobus monkeys at all times of the year during this survey. This absence of seasonality in infection is especially notable because of the extreme weather patterns typical of Kenya, where marked rainy and dry seasons occur. A second noteworthy observation is that the striking host specificity of the Cyclospora species initially described was confirmed in this survey. Baboons were only infected with C. papionis, vervet monkeys with C. cercopitheci, and colobus monkeys with C. colobi, despite geographic overlaps of both the monkey and parasite species and wide geographic distribution of each parasite and monkey host.     

Classification and distribution of large intestinal bacteria in nonhibernating and hibernating leopard frogs (Rana pipiens).

The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens. Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators. The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species. The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators. Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.     

Inheritance of enzymes and blood proteins in the leopard frog,Rana pipiens: Three linkage groups established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.The project was supported by Grant No. RR-00572 from the Division of Research Resources, National Institutes of Health. This paper is contribution No. C-87 from the Amphibian Facility, George W. Nace, Director.     

A 6 x 6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

A protocol was developed using 96-well plates and multichannel pipettes for serial dilutions, followed by drop plating on agar in a 6 x 6 format. This protocol permits simultaneous plating of six dilutions which greatly decreases the number of plates utilized thereby saving incubator space for organisms such as Campylobacter which require unique environmental conditions.     

Studies on two strains of Plasmodium cynomolgi in New World and Old World monkeys and mosquitoes

Infections that cause the Gombak and Smithsonian strains of Plasmodium cynomolgi were induced in Macaca mulatta, Aotus lemurinus griseimembra, Aotus nancymai, and Saimiri boliviensis monkeys. Transmission of the Gombak strain to Aotus spp. monkeys was obtained by the injection of sporozoites dissected from the salivary glands of experimentally infected Anopheles dirus and by the bites of infected An. dirus and Anopheles farauti mosquitoes. Two S. boliviensis monkeys were infected via the injection of sporozoites dissected from An. dirus. Prepatent periods in New World monkeys ranged from 14 to 44 days, with a median of 18 days. The Smithsonian strain was transmitted via sporozoites to 1 A. lemurinus griseimembra and 9 A. nancymai monkeys. Prepatent periods ranged from 12 to 31 days.     

Observations on the Vietnam Palo Alto strain of Plasmodium vivax in two species of Aotus monkeys

Thirty-three splenectomized Aotus lemurinus griseimembra monkeys with no previous experience with malaria were infected with the Vietnam Palo Alto strain of Plasmodium vivax. The median maximum parasite count was 280,000/microl. Nine splenectomized monkeys with previous infection with Plasmodium falciparum had median maximum parasite counts of 120,000/microl. Splenectomized Aotus nancymai monkeys supported infections at a lower level. Transmission via the bites of Anopheles dirus mosquitoes was obtained in a splenectomized A. lemurinus griseimembra, with a prepatent period of 31 days. It is estimated that between 1.5 x 10(8) and 1.6 x 10(9) parasites can be removed from an infected animal for molecular or diagnostic antigenic studies.     

Attenuation of Vesicular Stomatitis Virus Encephalitis through MicroRNA Targeting

Vesicular stomatitis virus (VSV) has long been regarded as a promising recombinant vaccine platform and oncolytic agent but has not yet been tested in humans because it causes encephalomyelitis in rodents and primates. Recent studies have shown that specific tropisms of several viruses could be eliminated by engineering microRNA target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. We therefore sought to determine whether microRNA targets could be engineered into VSV to ameliorate its neuropathogenicity. Using a panel of recombinant VSVs incorporating microRNA target sequences corresponding to neuron-specific or control microRNAs (in forward and reverse orientations), we tested viral replication kinetics in cell lines treated with microRNA mimics, neurotoxicity after direct intracerebral inoculation in mice, and antitumor efficacy. Compared to picornaviruses and adenoviruses, the engineered VSVs were relatively resistant to microRNA-mediated inhibition, but neurotoxicity could nevertheless be ameliorated significantly using this approach, without compromise to antitumor efficacy. Neurotoxicity was most profoundly reduced in a virus carrying four tandem copies of a neuronal mir125 target sequence inserted in the 3′-untranslated region of the viral polymerase (L) gene.Vesicular stomatitis virus (VSV) is a nonsegmented, negative-strand rhabdovirus widely used as a vaccine platform as well as an anticancer therapeutic. While VSV is predominantly a pathogen of livestock (34), it has a very broad species tropism. The cellular tropism of VSV is determined predominantly at postentry steps, since the G glycoprotein of the virus mediates entry into most tissues in nearly all animal species (10).Though viral entry can take place in nearly all cell types, in vivo models of VSV infection have revealed that the virus is highly sensitive to the innate immune response, limiting its pathogenesis (4). VSV is intensively responsive to type I interferon (IFN), as the double-stranded RNA (dsRNA)-dependent PKR (2), the downstream effector of pattern recognition receptors MyD88 (32), and other molecules mediate shutdown of viral translation and allow the adaptive immune response to clear the virus. The vulnerability of the virus to the type I IFN response, typically defective in many cancers, has been exploited to generate tumor-selective replication (49), such that the virus is now poised to enter phase I trials. However, the virus remains potently neurotoxic, causing lethal encephalitis not only in rodent models (7, 22, 53) but also in nonhuman primates (25).VSV very often infiltrates the central nervous system (CNS) through infection of the olfactory nerves (41). When administered intranasally, the virus replicates rapidly in the nasal epithelium and is transmitted to olfactory neurons, from which it then moves retrograde axonally to the brain and replicates robustly, causing neuropathogenesis. While intranasal inoculation does cause neuropathy in mice, neurotoxicity following viral administration also occurs when the virus is delivered intravascularly (47), intraperitoneally (42), and (not surprisingly) intracranially (13). Previously, other groups have modified the VSV genome to be more sensitive to cellular IFNs (49) and have actually encoded IFN in the virus (36). However, the former can result in attenuation of the virus, such that it has reduced anticancer potential, while the latter still results in lethal encephalitis (unpublished results). In order to mitigate the effects of VSV infection on the brain without perturbing the potent oncolytic activity of the virus, we utilized a microRNA (miRNA) targeting paradigm, whereby viral replication is restricted in the brain without altering the tropism of the virus for other tissues.To redirect the tissue tropism of anticancer therapeutics, we (26) and others (11, 14, 55) have previously exploited the tissue-specific expression of cellular miRNAs. miRNAs are ∼22-nucleotide (nt) regulatory RNAs that regulate a diverse and expansive array of cellular activities. Through recognition of sequence-complementary target elements, miRNAs can either translationally suppress or catalytically degrade both cellular (6) and viral (50) RNAs. We have determined that cellular miRNAs can potentially regulate numerous steps of a virus life cycle and that this regulation of the virus by endogenous miRNAs can then abrogate toxicities of replication-competent viruses (27; E. J. Kelly et al., unpublished data).miRNAs are known to be highly upregulated in many different tissues, including (but not limited to) muscle (40), lung (44), liver (15, 44), spleen (44, 46), and kidney (51). In addition, the brain has a number of upregulated miRNAs, with each different subtype of cell having a unique miRNA profile. miR-125 is highly upregulated in all cells in the brain (neurons, astrocytes, and glia cells), while miR-124 is found predominantly in neuronal cells (48). Glial cells and glioblastomas are thought to have decreased expression of miR-128 compared to neurons (17), while miR-134 is particularly abundant in dendrites of neurons in the hippocampus (43). In addition to these miRNAs, the tumor suppressor miRNA let-7 and miRs 9, 26, and 29 (51) are also found to be enriched in the brain, with expression varying not only between different cell types and regions of the brain but also temporally (48).MicroRNAs have previously been exploited to modulate the tissue tropism of nonreplicating lentiviral vectors (8, 9), as well as curbing known toxicities of replication-competent picornaviruses (5, 26), adenoviruses (11), herpes simplex virus 1 (33), and influenza A virus (39). In addition, a recombinant VSV encoding a tumor suppressor target was found to be responsive to sequence-complementary miRNAs in vitro, possibly by affecting expression of the matrix (M) protein (14), and evidence from Dicer-deficient mice suggests that endogenously expressed microRNA targets within the P and L genes of VSV could restrict enhanced pathogenicity of the virus (37). However, in vivo protection from neuropathogenesis by this means has not been demonstrated for VSV.Here we evaluate the efficiencies of different brain-specific miRNAs for shutting down gene expression and extensively characterize the ability of miRNA targeting to attenuate the neurotoxicity of vesicular stomatitis virus in vivo. We constructed and evaluated recombinant VSVs with miRNA target (miRT) insertions at different regions of the viral genome, with special focus upon those affecting viral L expression. In addition, we looked at the regulatory efficiency of different brain-specific miRNAs and the impact of miRT orientation on VSV replication and determined the impact of the virus on oncolytic activity in vivo.