Byron L. Blagburn

Cryptosporidium andersoni n. sp. (Apicomplexa: Cryptosporiidae) from Cattle, Bos taurus

Abstract A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 × 5.5 μm (range, 6.0–8.1 by 5.0–6.5 μm). The length to width ratio is 1.35 (range, 1.07–1.50). The colorless oocyst wall is < 1 μm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.

Molecular and phylogenetic characterisation of Cryptosporidium from birds.

Avian isolates of Cryptosporidium species from different geographic locations were sequenced at two loci, the 18S rRNA gene and the heat shock gene (HSP-70). Phylogenetic analysis of the sequence data provided support for the existence of a new avian species of Cryptosporidium infecting finches and a second species infecting a black duck. The identity of Cryptosporidium baileyi and Cryptosporidium meleagridis as valid species was confirmed. Also, C. baileyi was identified in a number of isolates from the brown quail extending the host range of this species.

Mechanical transmission of Toxoplasma gondii oocysts by dogs.

Two experiments were conducted to determine if dogs could mechanically transmit Toxoplasma gondii after ingesting cat feces or by rolling in cat feces containing oocysts. In the first experiment, two dogs were fed sporulated T. gondii oocysts; viable sporulated oocysts were present in dog feces for up to 2 days postinoculation (PI). Both dogs seroconverted to T. gondii but did not develop clinical signs of toxoplasmosis. In the second experiment, nonsporulated oocysts were placed on dog skin and fur, and fur clippings were bioassayed for T. gondii in mice. Oocysts did not sporulate on dog fur. The results of this study support the hypothesis that dogs may be involved in the mechanical transmission of T. gondii to humans.

A new Hepatozoon species from dogs : Description of the causative agent of canine Hepatozoonosis in North America

A new species of Adeleina, Hepatozoon americanum, is described from the skeletal muscle, cardiac muscle, visceral organs, and blood of dogs (Canis familiaris) in the Southern United States. The organism was previously identified as Hepatozoon canis (James, 1905) Wenyon, 1926; however, differences in clinical signs, histopathological and serological findings, gamont size, and ultrastructure define the new species of Hepatozoon. Attempts to transmit the protozoan from infected dogs to nymphal Rhipicephalus sanguineus ticks, the definitive host of H. canis, were not successful.

Prevalence of intestinal parasites in pet dogs in the United States.

To determine the national, regional, and age-related prevalence of intestinal parasites in dogs presenting to veterinarians in the United States, we reviewed the results of examination via zinc sulfate centrifugal flotation of 1,199,293 canine fecal samples submitted to Antech Diagnostics in 2006. The most commonly identified intestinal parasites were ascarids (2.2%), hookworms (2.5%), whipworms (1.2%), Giardia (4.0%), and Cystoisospora (4.4%). With the exception of whipworms, intestinal parasites were more commonly identified in dogs less than 6 months of age (29.6% positive) as compared to those greater than 1 year of age (6.1% positive) although infections with each parasite considered were identified in all age classes of dogs. Hookworm eggs were most commonly identified in fecal samples submitted from dogs from the South (4.0% positive), whereas ascarid eggs and Giardia cysts were most commonly seen in samples from dogs from the West (2.8% and 6.3% positive, respectively). When compared to previous data from shelter dogs, the prevalence of intestinal helminths, particularly ascarids and hookworms, was greatly suppressed in pet dogs in the southern United States (90-91% reduction) and much less so in dogs in the West (52-78% reduction), perhaps due in part to the routine year-round use of monthly anthelmintics effective at controlling both heartworm infection and intestinal helminths in dogs in the South. Taken together these data indicate that intestinal parasites remain a common, important finding in dogs presenting to veterinary practices although in most of the country infection rates in pet dogs appear to be greatly reduced from the level reported from dogs in animal shelters.

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.

Vertical transmission of Neospora caninum in dogs

Herein we report the development of a canine model to examine transplacental and/or transmammary transmission of Neospora caninum. Six pregnant bitches were experimentally infected with N. caninum on day 21 of gestation. One bitch successfully delivered a litter of 3 live pups. These pups showed slight proprioception deficits, increased muscle tone, and spasticity in both pelvic limbs; however, N. caninum was neither demonstrated by bioassay in cell culture nor seen in histological sections of tissues from these pups. The remaining 5 bitches aborted. Neospora caninum was isolated from tissues of 4 of the experimentally infected bitches and pups from 2 of the miscarried litters. Experimental infections of 2 litters of 5-day-old nursing pups produced variable results. Neospora caninum was isolated from 2 of 2 inoculated pups in 1 litter and 3 of 6 inoculated pups in the second litter.

Central Nervous System Neosporosis in a Foal

Cutaneous or subcutaneous cysts of all types are considered rare in cats. A literature search yielded no reports of cutaneous or subcutaneous dermoid cysts in cats. The structures reported here were histologically compatible with the description of the dorsal midline structures in dogs but, because they did not communicate with the spinal canal, these cysts posed no danger or potential danger to the animal from central nervous system infection. These structures were present in the flank instead of along the dorsal midline, possibly as a result of faulty embryologic fusion of adjacent dermatomes. The dermoid cyst in cat no. 2, considering its young age, probably was a congenital disorder; however, cat no. 1 was 10 years old at the time of presentation. It was not known how long the cyst in cat 1 had been present or if it had grown in size immediately prior to presentation. Dermoid cysts have been classified according to depth of penetration of the sinus. Class I cysts extend from the skin to the supraspinous ligament, class II cysts do not extend as deeply but are connected to the supraspinous ligament by a fibrous band, and class III cysts are similar to class II cysts but have no connecting band to the ligament. A fourth class has been proposed, in which the cyst extends to the spinal canal and is attached to the dura mater. This class is analogous to the pilonidal sinus of human beings, which usually occurs in the coccygeal region. The term pilonidal cyst, which by definition means any cyst containing a tuft of hair, is usually used synonymously with the term dermoid cyst in veterinary medicine. Perhaps 1 reason for the confusion over the term dermoid cyst is that many deep anomalous structures can have structural components of epithelium and can be loosely termed dermoid cysts by pathologists. A recent textbook has subclassified these cysts in an effort to more clearly define each type from a histologic standpoint. In this classification system, dermoid cysts are considered a type of follicular tumor. More widespread application of the classification system may be helpful to pathologists and clinicians for separating benign lesions from those with potential for serious complications. The surgical treatment of dermoid cysts is straightforward and involves careful dissection of the cyst. Although dermoid cysts are reported to involve only the skin or subcutaneous tissues, the cyst in cat no. 1 was found isolated between muscle layers of the flank and the sinus in cat no. 2 extended to the peritoneum. When excision of the cyst involves creation of an abdominal wall defect, care should be taken to ensure that anatomic closure of the defect is accomplished to avoid subsequent herniation of abdominal contents.

Correlation between loss of efficacy of macrocyclic lactone heartworm anthelmintics and P-glycoprotein genotype

Macrocyclic lactone (ML) molecules have been used for heartworm control for more than 25 years. However, in recent years, there have been reports of loss of efficacy of ML heartworm preventatives against Dirofilaria immitis in some locations in the United States. Macrocyclic lactone resistance is a common problem in nematode parasites of livestock, and more recently, evidence of ivermectin resistance has been reported in the human filarial nematode Onchocerca volvulus. In this study, four D. immitis sample groups from the United States with different treatment histories were investigated for evidence of ML-driven genetic selection. DNA from individual adult worms and microfilariae was amplified by polymerase chain reaction to investigate a gene encoding a P-glycoprotein, a protein class known to be involved in ML pharmacology. A significant correlation of a GG-GG genotype with ivermectin response phenotype was found. Moreover, a significant loss of heterozygosity was found in a low responder group; loss of heterozygosity is commonly seen in loci when a population has been under selection. Further studies are required to confirm ML resistance in heartworm populations. However, the genetic changes observed in this study may be useful as a marker to monitor for ML resistance in D. immitis.

Infection dynamics of Cryptosporidium parvum (Apicomplexa: Cryptosporiidae) in neonatal mice (Mus musculus).

maintained alive in a dechlorinated city water supply. Based on their size and immaturity, the hosts collected in the 1982 survey likely had not spent any time at sea. Anadromous fishes, such as eulachon (Thaleichthys pacificus (Richardson)) or possibly salmonids, that had returned to fresh water to spawn after a period of marine life were probably the source of the marine parasites found in the sturgeon. Semakula and Larkin (1968, loc. cit.) noted that eulachon form an important part of the diet of Fraser River white sturgeon. As mentioned above, Becker (1970, loc. cit.) offered a similar explanation for the occurrence of T. lindbergi in Columbia River white sturgeon. For reasons unexplainable at present, Corynosoma strumosum was found only in female sturgeon (9/21 examined), the difference in prevalence of infection between the sexes being statistically significant (Fishers exact probability test, P = 0.0083). There were no statistically significant host sex differences in prevalence of the other parasite species encountered. The present report represents the first on parasites of the Fraser River stock of white sturgeon, which is one of the major stocks of this North American sturgeon species. Voucher specimens of the parasites reported herein have been deposited in the National Museum of Natural Sciences, Invertebrate Collection (Parasites), Ottawa, Canada, and bear the following numbers: Crepidostomum auriculatum, NMIC(P) 1985-0115; Diphyllobothrium sp., NMIC(P) 1985-0116; Amphilina bipunctata, NMIC(P) 1985-0117; Corynosoma strumosum, NMIC(P) 1985-0118; Cystoopsis acipenseris, NMIC(P) 1985-0119; Anisakis simplex, NMIC(P) 1985-0120; undetermined nematode larvae, NMIC(P) 1985-0121. In addition, 4 specimens of C. auriculatum have been deposited in the Harold W. Manter Laboratory, University of Nebraska State Museum, Lincoln, Nebraska (No. 22983), and specimens of all species, except Diphyllobothrium sp. and A. bipunctata, are retained in the collection of the Pacific Biological Station. Specimens of A. bipunctata were given to Dr. G. Malmberg, University of Stockholm. We are pleased to acknowledge the assistance of Dr. Mary Hanson Pritchard and Dr. Janine Caira (Harold W. Manter Laboratory, University of Nebraska) in verifying the identification of C. auriculatum.