Application of quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types to examine associations between severity of anaemia and parasitaemia in bovine anaemia outbreaks

AIMS: To use quantitative PCR assays to detect Theileria orientalis Ikeda type in cattle presumed infected with T. orientalis, to examine the relationship between theilerial piroplasm count and haematocrit (HCT), and the relationship with quantification cycle threshold (Cq) values.

METHODS: Blood samples in EDTA (n=1,024), derived from herds affected by anaemia associated with T. orientalis infection (TABA) between April and October 2013, were submitted for testing using quantitative PCR (qPCR) assays for T. orientalis and Ikeda type. Nucleotide sequencing of the major piroplasm surface protein (MPSP) gene was performed on 16 samples to identify T. orientalis types. Blood smear and/or HCT results were supplied with most samples. For data analysis, the number of theilerial piroplasm per 1,000 erythrocytes counted was categorised as negative (0), low (1–9), moderate (10–100) or high (>100). HCT was categorised as severely anaemic (<0.15 L/L), mildly anaemic (0.15–0.24 L/L) or not anaemic (>0.24 L/L). Differences between categories in proportion of samples positive for Ikeda type or mean Cq value were examined using χ² tests or analysis of variance, respectively.

RESULTS: Of 1,022 samples containing amplifiable DNA, 916 (90%) were positive for T. orientalis and 789 (77%) were positive for Ikeda type. Nucleotide sequencing of MPSP amplicons also identified the presence of Chitose and Buffeli types in 11 samples without Ikeda. Ikeda was detected in a greater proportion of severely anaemic (288/302; 95%) than mildly anaemic (227/252; 90%) cattle (p=0.02). In non-anaemic cattle, 344/406 (85%) were positive for T. orientalis and 247/406 (60%) were positive for Ikeda type. In samples from cattle that were piroplasm-positive, a greater proportion of anaemic (483/505, 96%) than non-anaemic (211/307; 69%) cattle were positive for Ikeda type (p<0.001). In piroplasm-negative cattle, 20/37 (54%) anaemic and 25/78 (32%) non-anaemic cattle were Ikeda-positive (p<0.05). The distributions of Cq values differed between piroplasm count and HCT categories (p<0.001). Mean Cq differed between high and negative, and low piroplasm categories (p<0.001), but not between high and moderate categories (p=0.81), and differed between severely anaemic and mildly anaemic (p<0.001), and non-anaemic categories (p<0.001).

CONCLUSIONS: The Ikeda type was found in a high proportion of cattle during outbreaks of TABA in New Zealand. Analysis of Cq values suggested a relationship of Ikeda parasitaemia with severity of anaemia, but further investigation is required to better understand the role of parasitaemia in the pathogenesis of TABA.     

Severity and persistence of footrot in Merino sheep experimentally infected with a protease thermostable strain of Dichelobacter nodosus at five sites

Objective To test the hypothesis that ovine footrot associated with a thermostable protease strain of Dichelobacter nodosus undergoes self cure or is sustained as an annually recurring disease, depending on the environment.
Design and procedure Forty Merino sheep from a single blood line were infected with a protease thermostable strain of D nodosus a t each of five sites in Western Australia. Footrot lesions and microscopic evidence of D nodosus were recorded every fortnight for 2.5 years, supplemented by laboratory culture. Rainfall, soil and air temperature, pasture quantity and composition and soil types were also recorded. Flocks that apparently self cured were relocated to a more favourable site for footrot in the final spring season.
Results The maximum prevalence of feet with clinical footrot lesions was 80.6, 1.3, 14.4, 3.8 and 88.1% at the five sites. Severe footrot occurred for three consecutive spring seasons at one site that had clay loam soil and at least 3500 kg/ha total pasture dry matter annually. However, the infection was asymptomatic for up to 10 weeks between outbreaks. D nodosus was isolated from flocks for 2.5 years at only two sites, although there was microscopic evidence of the organism at other sites in the final year. A thermolabile variant (strain U6) of D nodosus was isolated from the two sites where footrot persisted.
Conclusion Depending on time and location, ovine footrot induced by a protease thermostable strain of D nodosus either self cured or persisted as annual outbreaks interspersed with periods of asymptomatic infection.     

Clinical evaluation of protein electrophoresis of cerebral spinal fluid

Protein fractions of cerebral spinal fluid samples were analyzed in an effort to find a reliable, minimally invasive and cost effective method of diagnosing cervical vertebral myelopathy. There were six separate protein fractions analyzed by electrophoresis. In this study we specifically looked for a post beta spike in horses with spinal cord compression. There was no correlation in our study between horses with a post beta spike and CVM as diagnosed by a myelogram.     

Fatty acid profiles of crop contents of free-living psittacine nestlings and of commercial hand-feeding formulas

Research on psittacine nutrition is limited, and nestling requirements are poorly understood. This study analysed fatty acid (FA) profiles of crop contents of free-living scarlet macaws (Ara macao, n = 18), red-and-green macaws (Ara chloropterus, n = 5), Cuban parrots (Amazona leucocephala bahamensis, n = 27), lilac-crowned Amazons (Amazona finschi, n = 33) and thick-billed parrots (Rhynchopsitta pachyrhyncha, n = 32). The same analysis was carried out on 15 commercial parrot hand-feeding formulas. The mean FA concentration of the crop samples of each species ranged from 15% to 53% DM for crop samples and ranged from 6% to 22% for hand-feeding formulas. Long-chain FA represented over 92% of all FA in the crop samples and over 81% of all FA in the commercial formulas. Parrot species shared similarities in saturation profiles of crop samples, ranging between 13%–29% saturated fatty acids (SFA), 12%–40% monounsaturated fatty acids (MUFA) and 39%–58% polyunsaturated fatty acids (PUFA). All studied psittacines, except for the red-and-green macaw, were within the range of values for hand-rearing formulas. Palmitic acid was the most common SFA in scarlet macaws, red-and-green macaws, Cuban parrot, thick-billed parrot and in all but one commercial formula. Palmitic and stearic acids dominated the SFA in the samples of the Lilac-crowned Amazon. Oleic acid was the most common MUFA in all hand-feeding formulas as well as in the crop samples, except for the lilac-crowned amazon and the thick-billed parrot where vaccenic acid dominated. Linoleic acid was by far the most common PUFA found in the crop samples as well as in the hand-feeding formulas. PUFA were largely dominated by the n6 family, both in the crop samples and the formulas. The data presented on nestling diets of free-living parrot species provide a foundation for future researchers to test whether increasing FA concentration in hand-feeding formulas improves nestling development or if species-specific formulas will be advantageous.     

The use of hand-raised psittacines for reintroduction: a case study of scarlet macaws (Ara macao) in Peru and Costa Rica

This study reports on three scarlet macaw (Ara macao) reintroduction projects using hand-raised birds in Peru and Costa Rica. The habitats at the release sites ranged from pristine tropical forest to forest fragments in an agricultural matrix. The combined first-year survival was 74% and the annual post first-year survival was 96%. Survival rates were very high despite a wide range in predator communities. Number of birds released explained 70% of the variation in survival with birds from larger releases having higher survival rates. Behavioral evidence suggests that birds established at the site facilitated survival of later releases. Breeding attempts were recorded at all three sites and hand-raised birds with wild mates successfully fledged young in Peru. Supplemental feeding post-release played an important role in keeping the birds near the release site and facilitating social interactions. This work shows that properly socialized hand-raised macaws can survive and breed in the wild but that ex-pets are not good release candidates.     

Effect of Insemination–Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.     

Immunological detection of cardiac glycosides in plants

Summary Australian native and introduced plants were examined, using digoxin immunoassays, to detect the presence of cross-reacting cardiac glycosides. These compounds were found in 27 species from 20 genera. The assay technique can also be used on serum samples to confirm cardiac glycoside ingestion.