This material is freely available for teaching and other non-commercial uses. Milton M. McAllister milton.mcallister@adelaide.edu.au The article “Diagnosis and control of bovine neosporosis” is available at this link for free download for a limited time, until 7 August 2016 http://authors.elsevier.com/a/1TE2f_H22n97eD

Fig. 1, from Diagnosis and Control of Bovine Neosporosis Fig. 1, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016. Fig. 1. Neurologic impairment in calves infected with Neospora caninum. The beef calf at left was born following a neosporosis abortion outbreak. It was undersized, had weak hindlimbs, and a conscious proprioceptive deficit that is here demonstrated by the dorsal placement of the left rear hoof. The dairy calf at right was unable to stand, maintain sternal recumbency, or elevate its head.

Neospora caninum protozoal life cycle Latent microscopic cysts in tissues contain bradyzoites tissues ingested Asexual and sexual reproduction Oocysts in canine feces (10-11 µm) exposure to air Oocysts sporulate and disseminate in dust and water Tachyzoites replicate intracellularly and spread to other cells defecation (N. caninum immunohistochemistry and modified Giemsa impression smears of canine intestine) (immunofluorescent stain of cultured organisms) oocysts ingested by Intermediate Host immune response controls proliferation by Definitive Host (contrast illumination) Fig. 2. Neospora caninum life stages. (upper left) Photomicrograph of a squash preparation showing a spherical, thick-walled cyst containing numerous bradyzoites; this latent stage survives in tissues for prolonged periods. (upper right) Asexual and sexual reproductive forms of the parasite that occur in canine intestine after ingestion of infected tissues. (right) Egg-like oocyst, shed in canine feces. (bottom right) Sporocysts containing sporozoites have developed within an oocyst, which is now infectious. (bottom left)Upon ingestion of infectious oocysts by an intermediate host animal, sporozoites are liberated by digestive juices, invade host cells, and become rapidly dividing tachyzoites; if unchecked by the immune response, tachyzoites may cause clinical disease. Otherwise, they convert to latent bradyzoites and the cycle is completed. Fig. 2, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016.

Transmission of Neospora caninum. Fig. 3. Transmission of Neospora caninum. Cattle may become infected “horizontally” by ingestion of oocysts at any time of life (not only when pregnant). Congenital infection may result if infection is first acquired by a dam during pregnancy (exogenous transplacental transmission), or by reactivation of organisms from a latently infected dam (endogenous transplacental transmission). Artist: Kerry Helms, University of Illinois. Fig. 3, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016.

Relation between the time of maternal exposure to Neospora caninum oocysts and the outcome of pregnancy in naive cattle Time of Exposure First trimester Second trimester Third trimester Fetuses on this side of the solid curve are infected, but are clinically normal if above the dotted curve Fetuses below both curves are aborted or impaired Fetuses on this side of the curve have not acquired infection No fetal immune system Maturing fetal immune system Pathogenicity of Transplacental to Fetus Transmission Low High Likelihood Fig. 4. Relation between the time of maternal exposure to Neospora caninum oocysts and the outcome of pregnancy in naive cattle. Fig. 4, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016.

A D C B Styrofoam container Case sheet in bag protects clerical staff. Information assists diagnostics and reduces unnecessary phone calls for clarification. Cold pack Absorbent packing Double-bagged specimens keep items sterile and prevent leaks. Fig. 5. Submitting specimens for abortion diagnostics. (A)Wet specimens of any type should be packaged within 2 containers, an inner primary and an outer secondary container, with absorbent material in between. Here, absorbent material is placed in the bottom of a biological specimen bag (not a zip-closing sandwich bag) to provide reliable secondary containment. (B) Tightly closed primary specimen containers are suitable for formalin specimens or fresh tissues. (C) Vacuum serum tubes are suitable sterile containers to hold liquid specimens including abomasal fluid, fetal fluids from thorax or abdomen, and serum from the aborting dam. (D) A package ready to be sealed and shipped. Fig. 5, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016.

F G E A B C D Fig. 6. Potential contamination of feedstuffs with Neospora caninum oocysts and dissemination in mixed rations. Representative dairy (A–E) and beef (F, G) farms are depicted that each had a recent neosporosis outbreak. Clockwise from upper right: (A) Above ground ensiling practices increase the opportunity for rodents to frequent or inhabit the silage, especially at the leading edge and around the sides, where discoloration reveals that anaerobic conditions have not been adequately maintained. When examined, rats and mice were flushed out of the silage pile. Dogs and other canids are attracted both to open silage and to rodents. (B) Examples of ration components piled in open bays or outside at a large dairy farm. (C) Various components of the mixed ration. (D) Use of a front-end loader and mixer wagon to prepare a TMR. If any component is contaminated with Neospora oocysts (or other orally transmitted pathogens such as Leptospira or Salmonella), then it will become distributed throughout the TMR. (E) Feeding the TMR to a pen of dairy cows. Potential exists to deliver pathogens to many animals at a time. (F) This outdoor silage pile on a beef farm was not covered, and animal tracks and scats of many types were present, potentially increasing attractiveness to dogs and coyotes. (G) Provision of mixed silage and chopped hay to the beef cow herd, which were periodically brought in from winter pasture for supplemental feeding. Fig. 6, from Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister MM, 2016.

Combined 1744 / 2762 63% Citation Year Location Table 2. Endogenous transplacental transmission rates of Neospora caninum in dairy cattle. Studies were included if they tested the offspring of at least 100 seropositive dams. These calculations have not been adjusted to account for horizontal transmission, which could cause upward skewing. Citation Year Location Seropositive offspring / Seropositive dams Simple calculation of endogenous transmission rate [66] 2004 Ontario 252 / 619 41% [42] 2007 Netherlands 325 / 526 62% [67] 2003 363 / 500 73% [68] 2005 Sweden 316 / 369 86% [46] Costa Rica 168 / 285 59% [45] 2001 163 / 204 80% [69] 2000 Quebec 64 / 144 44% [70] 1996 California 93 / 115 81%   Combined 1744 / 2762 63% modified from Table 2 in: Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister, Milton M. 32:443-463. 2016.

were administered to cows Table 1. Results of administration of Neospora caninum oocysts to pregnant cows. Data were compiled from the only three published experiments of this type. Day of gestation that oocysts were administered to cows   70 120 – 130 162 - 210 Number of cows that became infected and were followed to parturition 7 9 Number of offspring that became infected 3 6 Proportion of infected cows having infected offspring 0.00 0.33 0.86 Number of cows having abortions or stillbirths Number of aborted fetuses or stillborn calves that were infected - Proportion of infected offspring that were aborted or stillborn 1.00 Table 1 in: Diagnosis and Control of Bovine Neosporosis. Veterinary Clinics of North America: Food Animal Practice. McAllister, Milton M. 32:443-463. 2016.

Virus Protozoan Bacteria Phylum Apicomplexa Apical complex Photo from: Duane’s Foundations of Clinical Opthalmology, Chapter 76, Ocular Toxoplasmosis, Holland & O’Connor http://www.oculist.net/downaton502/prof/ebook/duanes/pages/v8/ch076/002f.html mitochondria nucleus without organelles without cytoplasm

www.ottersandbutterflies.co.uk wikipedia Stage changes of insects are similar to stage changes that occur in Apicomplexan parasites

(like dividing caterpillars) Stages of Neospora caninum or Toxoplasma gondii Tachyzoites (like dividing caterpillars) Bradyzoites in cysts (crysallis in cocoon) Sexual forms (butterflies) Oocysts (eggs)

Tachyzoites - multiply inside of host cells - may cause disease

Tachyzoites in cell culture (fluorescent antibody label) Necrotic focus rimmed by infiltrating leukocytes in the brain of an aborted calf Immunohistochemistry demonstrating tachyzoites (black) within a cerebral lesion McAllister McAllister Tachyzoites in cell culture (fluorescent antibody label) McAllister

Typical lesions in aborted bovine fetus: nonsuppurative inflammation in epicardium, myocardium, muscle, and brain

Bradyzoites “hibernate” within cells in muscle and CNS infect the next animal by carnivorism

A cyst containing bradyzoites. Squash prep from the brain of a laboratory mouse. McAllister

Oocysts shed in canine feces resistant to environmental degradation orally infectious to intermediate hosts O2 Nonsporulated oocyst diploid Sporulated oocyst haploid

This is the only diploid stage in the life cycle 10 μM Nonsporulated oocyst This is the only diploid stage in the life cycle Sporulated oocyst Two sporocytes inside of the oocyst, each containing four haploid sporozoites Sporulated oocyst photo is from: McAllister MM., Dubey JP, Lindsay DS, Jolley WR, Wills RA, McGuire AM. Dogs are definitive hosts of Neospora caninum. Int J Parasitol 28:1473-1478, 1998. Neospora caninum

Photo from: Gondim L. F. P. et al Photo from: Gondim L.F.P. et al., Transplacental transmission and abortion in cows administered Neospora caninum oocysts. J Parasitol 90, 1394-1400, 2004