Jennifer A. Spencer

Prevalence of antibodies to Neospora Caninum in dogs

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.

Experimental infection of cats (Felis catus) with Tritrichomonas foetus isolated from cattle

Tritrichomonas foetus is recognized as the causative agent of venereal trichomoniasis in cattle. It is characterized by embryonic and early fetal death and post-coital pyometra, and feline trichomoniasis, manifest as chronic, large bowel diarrhea. Many of the infected cats are less than 2 years old and specific routes of transmission remain unknown. We recently demonstrated that feline isolates of T. foetus can successfully infect heifers, resulting in pathologic changes similar, but not identical to those previously reported as representative of bovine trichomoniasis. In this study, we experimentally infected six cats less than 1 year of age with a bovine (D-1) isolate of T. foetus and one cat with a feline (AUTf-1) isolate of T. foetus. Within 2 weeks, the cat infected with the feline (AUTf-1) isolate was culture positive for trichomonads in weekly fecal samples. At the end of 5 weeks, only one cat infected with the bovine (D-1) isolate was fecal culture positive for trichomonads. At necropsy, the intestine of each cat was removed and divided into five sections (ileum, cecum, anterior, medial and posterior colon). Contents from each section were collected and cultured. The cat infected with the feline (AUTf-1) isolate was culture positive in the ileum, cecum, medial and posterior colon. Two cats infected with the bovine (D-1) isolate were culture positive in the cecum only. Additionally, each intestinal section was submitted to a pathologist for histopathological examination. The combined results indicate that there are demonstrable differences between the feline (AUTf-1) and bovine (D-1) isolates regarding their infectivity in cats.

EXPERIMENTAL INFECTION OF CATTLE WITH A FELINE ISOLATE OF TRITRICHOMONAS FOETUS

Tritrichomonas foetus is the causative agent of bovine trichomoniasis, a sexually transmitted disease in cattle that can result in large profit losses for cattle producers. Increasing reports have suggested that T. foetus is also the causative agent of large-bowel diarrhea in cats. To determine if the trichomonads recovered from the reproductive tract of cattle and the large intestine of cats can thrive in the same host, 2 groups of virgin Angus heifers were inoculated with T. foetus. The first group of heifers was inoculated with a bovine T. foetus isolate cultured from a naturally infected cow, and heifers in the second group were inoculated with T. foetus organisms cultured from the feces of a naturally infected cat. Over an 11-wk period, vaginal, cervical, and uterine mucus samples were analyzed, along with a single transcervical uterine biopsy sample, to determine organism and disease presence. The mucus and biopsy samples collected from each group indicate that the disease caused by feline and bovine isolates of T. foetus are comparable, but not identical.

SEROPREVALENCE OF NEOSPORA CANINUM AND TOXOPLASMA GONDII IN CAPTIVE AND FREE-RANGING NONDOMESTIC FELIDS IN THE UNITED STATES

Abstract Seven serum samples of 101 samples from nondomestic, captive and free-ranging felids from the United States were indirect fluorescent antibody positive for antibodies to Neospora caninum, whereas 44 samples were positive for antibodies to T. gondii. Although none of the captive animals displayed clinical signs of disease, nondomestic felids in the United States have been exposed to, and are likely infected with, N. caninum and T. gondii. This may have serious implications for zoological gardens exhibiting susceptible animals, such as kangaroos, close to felids.

Disseminated Toxoplasmosis in a Captive Ring-Tailed Lemur (Lemur catta)

A 3-yr-old secundiparous female ring-tailed lemur presented to the Auburn University Small Animal Clinic with signs of dyspnea, lethargy, and anorexia. The animal died before she could be examined, and a full necropsy was immediately performed. Provisional necropsy findings included moderate pneumonia and hepatopathy. Acute interstitial pneumonia and focal hepatocellular necrosis were confirmed histologically. Lung impression smears, histopathology, electron microscopy, immunohistochemistry, and tissue culture isolation resulted in a diagnosis of acute disseminated Toxoplasma gondii infection, which was confirmed by polymerase chain reaction. The isolate of T. gondii was avirulent for mice and was named AU Tg1 and genetically is type II. The source of the infection remains unclear, but speculation suggests contaminated fruit or blackbirds (Passeriformes: Icteridae) acting as transport hosts for oocysts from nondomestic felids and feral cats on the property.

Evaluation of permethrin and imidacloprid for prevention of Borrelia burgdorferi transmission from blacklegged ticks (Ixodes scapularis) to Borrelia burgdorferi-free dogs

Lyme borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi. The vector tick, Ixodes scapularis is infected during larval tick feeding. Transstadial transmission occurs in the tick, which may result in infection of a mammalian host during feeding as a nymph or adult. The disease is of significance as both dogs and people are susceptible to Lyme borreliosis. The aim of this study was to evaluate the ability of a product containing imidacloprid and permethrin (K-9 AdvantixTM) to prevent transmission of B. burgdorferi to dogs. Adult Beagle dogs confirmed to be free from B. burgdorferi exposure via IFA testing, were randomly assigned to two groups (treated and nontreated control) of 8 animals each and housed separately in a BL-2 facility. K-9 AdvantixTM was applied to one or several sites the dorsal midline of all 8 treated dogs. Field caught adult Ixodes scapularis with an average infectivity rate of 57.6% were used as source of B. burgdorferi organisms. One week post treatment 100 ticks per dog were placed on the backs of the dogs and encouraged into the hair coat. Serum antibody titers to B. burgdorferi were obtained for weeks 2 through 13. By week 6 all nontreated control dogs had seroconverted and all treated dogs continued to remain antibody negative. There was total agreement between the ELISA and IFA antibody results. Skin punch biopsies were obtained for PCR testing and these results are still pending. The results of this study show that K-9 AdvantixTM is effective 7 days after treatment in the prevention of transmission of Lyme borreliosis from infected I. scapularis ticks to dogs.

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons, Procyon lotor, from the United States.

Equine protozoal myeloencephalitis (EPM) is the most important protozoal disease of horses in North America and it is caused by Sarcocystis neurona. Natural cases of encephalitis due to S. neurona have been reported in raccoons, Procyon lotor. We examined 99 raccoons for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. Raccoons originated in Florida (N=24, collected in 1996), New Jersey (N=25, collected in 1993), Pennsylvania (N=25, collected in 1999), and Massachusetts (N=25, collected in 1993 and 1994). We found that 58 (58.6%) of the 99 raccoons were positive for antibodies to S. neurona using the SAT; 44 of 99 raccoons (44%) had titers of > or =1:500. This prevalence is similar to the reported seroprevalence of 33-60% for S. neurona antibodies in horses from the United States using the Western blot test.

Inoculation of Sarcocystis neurona merozoites into the central nervous system of horses.

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. A horse model of EPM is needed to test the efficacy of chemotherapeutic agents and potential vaccines. Five horses that were negative for antibodies to S. neurona in their serum and cerebrospinal fluid (CSF) were injected in the subarachnoid space with living merozoites of the SN2 isolate of S. neurona. None of the horses developed clinical disease or died over a 132-day observation period. All five horses developed antibodies to S. neurona in their CSF and serum 3-4 weeks after injection. Two of the horses were examined at necropsy and no parasite induced lesions were observed in their tissues and no parasites were recovered from portions of their spinal cords inoculated on to cell cultures. Results of this study demonstrate that merozoites of the SN2 isolate of S. neurona will induce seroconversion but not clinical disease when inoculated directly into the CSF of nonimmune horses.

Prevalence of Agglutinating Antibodies to Neospora caninum in Raccoons, Procyon lotor

Neospora caninum is an apicomplexan parasite that causes neonatal neuromuscular disease in dogs and abortions in cattle. Dogs are the only proven definitive host. Little is known about the prevalence of antibodies to this parasite in wildlife. Sera from 99 raccoons (Procyon lotor) were examined for agglutinating antibodies to N. caninum using the modified agglutination test employing formalin-fixed tachyzoites as antigen. Raccoons originated in Florida (n = 24, collected in 1996), New Jersey (n = 25, collected in 1993), Pennsylvania (n = 25, collected in 1999), and Massachusetts (n = 25, collected in 1993 and 1994). Ten (10%) had antibodies to N. caninum; 9 had titers of 1:50, and 1 (1%) had a titer of 1:100. The present study indicates that raccoons have minimal exposure to N. caninum. The sera were also tested for agglutinating antibodies to Toxoplasma gondii and 46 (46%) were positive; 16 had titers of 1:50, 8 had titers of 1:100, and 22 had titers of ≥1:500.

Cytokine gene expression in response to SnSAG1 in horses with equine protozoal myeloencephalitis.

ABSTRACT Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to induce immunosuppression towards parasite-derived antigens as parasite-specific responses are decreased. Isolated peripheral blood lymphocytes from 21 EPM (cerebrospinal fluid [CSF] Western blot)-negative horses with no clinical signs and 21 horses with clinical signs of EPM (CSF Western blot positive) were cocultured with SnSAG1 for 48 and 72 h, and the effect on cytokine production was investigated by means of reverse transcriptase PCR. Cytokines assayed include gamma interferon (IFN-γ), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, and IL-6. β-Actin was used as the housekeeping gene. A Wilcoxon signed-rank test of the findings indicated that there was a statistically significant decrease in IFN-γ production after 48 h in culture for samples from horses with clinical disease. There was also a statistically significant increase in IL-4 production after 72 h in culture for samples from horses with EPM. These results further support the notion that this parasite is able to subvert the immune system in horses with clinical disease.