Mark C. Jenkins

Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences

Complete 18S ribosomal RNA gene sequences were determined for 8 Eimeria species of chickens and for Eimeria bovis of cattle. Sequences were aligned with each other and with sequences from 2 Sarcocystis spp., Toxoplasma gondii, Neospora caninum, and 4 Cryptosporidium spp. Aligned sequences were analyzed by maximum parsimony to infer evolutionary relationships among the avian Eimeria species. Eimecia bovis was found to be the sister taxon to the 8 Eimeria species infecting chickens. Within the avian Eimeria species, E. necatrix and E. tenella were sister taxa: this clade attached basally to the other chicken coccidia. The remaining Eimeria spp. formed 3 clades that correlated with similarities based on oocyst size and shape. Eimeria mitis and Eimeria mivati (small, near spherical oocysts) formed the next most basal clade followed by a clade comprising Eimeria praecox. Eimeria maxima, and Eimeria brumetti (large, oval oocysts), which was the sister group to Eimeria acervulina (small, oval oocysts). The 4 clades of avian Eimeria species were strongly supported in a bootstrap analysis. Basal rooting of E. necatrix and E. tenella between E. bovis and the remaining Eimeria species and the apparent absence of coccidia that infect the ceca of jungle fowl all suggest that E. necatrix and E. tenella may have arisen from a host switch, perhaps from the North American turkey, Meleagris gallopavo.

Redescription of Neospora caninum and its differentiation from related coccidia

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums.

Oral infection of calves with Neospora caninum oocysts from dogs: humoral and cellular immune responses.

Neospora caninum has been identified as a major cause of abortion in cattle in a number of countries throughout the world. Until the recent demonstration that dogs can serve as a definitive host of this parasite, it was not possible to study the infection in cattle orally exposed to oocysts. The aim of this study was to investigate the potential of N. caninum oocysts to infect calves, and to define initial immune responses that arise after oral infection. Seven calves were fed approximately 10(4)-10(5) N. caninum oocysts, three calves served as uninfected controls. Before infection, all calves were serologically negative for anti-Neospora antibodies and the calves were non-reactive to Neospora antigen in an in vitro lymphocyte proliferation assay. Peripheral blood lymphocytes from inoculated calves were able to mount in vitro proliferative responses to crude N. caninum antigen extract as early as 1 week p.i. Within 2 and 4 weeks p.i., Neospora-specific IgG1 and IgG2 antibodies were detected by IFAT and ELISA in serum from infected calves but not from sham-infected calves. The continued presence of reactive cells in the blood, spleen and mesenteric, inguinal, bronchial lymph nodes was seen as late as 2.5 months p.i., and parasite DNA was detected in the brain and spinal cord of the infected animals by PCR, indicating that the cattle were infected by oral inoculation of N. caninum oocysts collected from dogs, and that the animals were systematically sensitised by parasite antigen.

Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures.

Oocysts of Cryptosporidium parvum obtained from calves were cleaned of fecal debris by density gradient centrifugation and suspended in deionized water in microcentrifuge tubes. The tubes were placed in circulating water baths at temperatures of -10, -5, 0, 5, 10, 15, 20, 25, 30, or 35 C, and 2 tubes were removed from each water bath 1, 2, 4, 8, 12, 16, 20, and 24 wk later. Oocysts from 1 tube were administered at the rate of 1.5 x 10(5) oocysts per mouse to 2 litters of neonatal BALB/c mice and were considered infective when developmental stages were found in histologic sections of mouse gut and/or a positive polymerase chain reaction (PCR) was obtained for C. parvum DNA in mouse ileum. The second tube was held at -70 C until tubes from all time periods were available, then oocysts within the tubes were assayed for amylopectin concentration. Oocysts held at -10 C were infectious up to 1 wk of storage, and those held at -5 C were infectious up to 8 wk of storage, as determined by PCR but not histology. Oocysts held at 0, 5, 10, 15, and 20 C were still infectious after 24 wk of storage. By microscopic examination of mouse tissue, oocysts held at 20 C infected only 1 of 10 mice after 24 wk of storage, and the number of developmental stages began declining after 4 wk of storage; those held at 25 and 30 C each produced infections up to 12 wk after storage in 1 of 10 mice with reduced numbers of developmental stages beginning 4 wk after storage. Those held at 35 C produced light infections in 2 of 10 mice only up to 1 wk of storage. Amylopectin concentration decreased with increasing length of storage time or temperature. These findings provide a guide for estimating the potential duration of oocyst infectivity within a wide range of environmental temperatures and demonstrate the relationship between amylopectin concentration and infectivity.

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.

A recombinant Eimeria protein inducing interferon-γ production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis.

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.

Canine neosporosis: clinical signs, diagnosis, treatment and isolation of Neospora caninum in mice and cell culture

Clinical signs, diagnosis, treatment and isolation of Neospora caninum from two littermate dogs are described. Three of six pups from a Labrador bitch developed paralysis. Neosporosis was diagnosed ante mortem by serological examination in two of the affected pups. At necropsy, tissue cysts were seen in unstained smears and in histologic sections of their brains. Tissue cysts were often thin-walled (approximately 1 micron) but antigenically and ultrastructurally identified as N. caninum. Furthermore, N. caninum (isolates NC-4, NC-5) was isolated in mice and in cell cultures inoculated with neural tissues of these two dogs. Serological diagnosis of neosporosis using a variety of tests is discussed.

Prevention of vertical transfer of Neospora caninum in BALB/c mice by vaccination.

Neosporosis is an important cause of abortion and neonatal morbidity in dairy cattle. The disease is caused by Neospora caninum, an intracellular protozoan parasite. In this report, we describe the use of a mouse model in the preliminary evaluation of vaccination as a means to prevent vertical transfer of N. caninum. Parasites present in the tissues of the offspring were detected using an N. caninum-specific polymerase chain reaction assay. Immunization of dams with a single inoculation of a crude lysate of N. caninum tachyzoites appeared to induce complete protection against infection of the offspring.

The genus Hammondia is paraphyletic

The phylogenetic relationships amongst Hammondia, Neospora and Toxoplasma were investigated by DNA sequence comparisons of the D2/D3 domain of the large subunit ribosomal DNA and the internal transcribed spacer 1. The results obtained allow us to reject the hypothesis that N. caninum and H. heydorni are the same species and show that Hammondia hammondi is probably the sister taxon to Toxoplasma gondii.

ANTIBODY RESPONSES OF COWS DURING AN OUTBREAK OF NEOSPOROSIS EVALUATED BY INDIRECT FLUORESCENT ANTIBODY TEST AND DIFFERENT ENZYME-LINKED IMMUNOSORBENT ASSAYS

Serum samples from 70 (33 aborting and 37 non-aborting) dairy cows from a herd in California were analyzed for Neospora caninum antibodies in different laboratories by various serologic assays including enzyme-linked immunosorbent assay (ELISA) with recombinant antigens (Nc4.1 and Nc14.1), kinetic ELISA, whole tachyzoite lysate ELISA, immunostimulating complex (iscom) ELISA, antigen capture competitive inhibition ELISA, and by the indirect fluorescent antibody test. Eighteen percent of pregnant cows in this herd had aborted within 2 mo of the index case. All 70 cows had antibodies to N. caninum by at least 1 of the tests. Antibody levels to N. caninum in aborting cows as a group were higher than in nonaborting cows. However, it was concluded that no serological test could be used to establish definitively that N. caninum caused the abortion in an individual cow.