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    A comparative study between milk- and serum-based antibody detection assays for Johne's disease in New Zealand dairy cattle
    (Elsevier B.V., 2025-08-27) Venkatesh KM; Lopez-Villalobos N; Gupta SK; Udy GB; Laven R; Chiu S-J; Bugde P; Furuya Y; Dukkipati VSR
    Dairy cattle are affected by Johne's disease. It is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Suboptimal diagnostic tests add more to the productivity loss resulting from this disease. Agreement between and within different commercial kits is crucial in the decision-making process of disease surveillance programmes. This study compared two ELISAs, that is, Johne's disease commercial antibody detection kits (A and B), using milk and serum samples from New Zealand dairy cattle. These results were also compared with a subset of faecal PCR results. Five scenarios were considered for the comparison of ELISA tests. The point estimates of kappa coefficients (k) between the serum (0.84–0.94) assays were higher than the milk assays (0.59–0.82). The point estimates of kappa coefficients between serum and milk ELISA outcomes were higher for kit B (k = 0.79–0.86) than for kit A (k = 0.55–0.79). The point estimates of kappa coefficients between the ELISA and faecal PCR outcomes varied between 0.43 and 0.74. ELISA tests had point estimates of sensitivity ranging from 0.67 to 0.88 and specificity from 0.62 to 0.93, relative to the faecal PCR test. Results suggest that serum provides a better choice of sample type when both commercial kits A and B are used for Johne's disease surveillance of dairy cattle in New Zealand. Milk assays can be cost-effective to diagnose MAP-positive animals; kit B can be best suited for New Zealand conditions, provided the repeatability of the results is validated.
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    Comparison of enzyme-immunoassay of oestrone sulphate in milk with rectal palpation, ultrasonography and farmers' observation for pregnancy diagnosis in seasonal dairy herds in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master in Veterinary Science at Massey University
    (Massey University, 2001) Mueller, Karin
    A total of 2139 cows in six commercial, spring-calving New Zealand dairy herds were examined for pregnancy by enzyme-immunoassay of oestrone sulphate in milk, rectal palpation and real-time ultrasonography at 137 to 180 days after the start of mating. The gold standard was based on calving records, observed events such as abortion, or examination of the reproductive tract after slaughter. Sensitivity was 81.8%, 100.0% and 99.9%, and specificity was 81.0%, 91.4% and 90.9% for oestrone sulphate, rectal palpation and ultrasonography, respectively. Oestrone sulphate sensitivity increased in a linear fashion with advancing stage of gestation and reached 96.8% for cows at least 120 days pregnant. Sensitivity and specificity of oestrone sulphate were significantly lower than those of the other two methods were significant (p=0.0001). In seven additional herds with a total of 967 animals, a pregnancy diagnosis was obtained by oestrone sulphate and farmers' observation. Sensitivity and specificity for these two methods were significantly different at 85.4% vs. 98.6% (p=0.0001), and 80.4% vs. 66.7% (p<0.002), respectively. The sensitivity of oestrone sulphate increased and the specificity of farmers' observation decreased with advancing stage of pregnancy. Using a partial farm budget, the cost of pregnancy diagnosis by oestrone sulphate was established as NZ$ 6.54 per cow compared to NZ$ 4.34 for rectal palpation and NZ$ 4.60 for ultrasonography. Compared to farmers' observation, oestrone sulphate was more expensive at NZ$ 6.63 vs. NZ$ 6.53 per cow.
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    Characterisation of a secreted immunogenic protein, phase-1 flagellin (FliC) of Salmonella enterica subspecies enterica Brandenburg : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Perera, Kalyani
    Cell-envelope associated and secreted proteins of Salmonella are integral for host-pathogen interactions, and for the induction of protective immune responses. An array of exported proteins of S. Brandenburg was identified through constructing an expression library using alkaline phosphatase gene technology. A partial digest of S. Brandenburg strain S59 was cloned into the vector pJEM11, and expressed in E. coli. The DNA inserts from randomly selected alkaline phosphatase positive clones were sequenced, and the sequences were analysed using public databases to find the ones that may play a role in host immune cell activation. The phase-1 flagellin (fliC) gene identified from an alkaline phosphatase positive phenotype was chosen for further studies. The complete nucleic acid sequence of the fliC gene was obtained by PCR amplification. The complete ORF, part of the variable region (V456) and region IV (V4) of the fliC gene were cloned into the pET14b vector for the expression of N-terminal histidine-tagged fusion proteins. The proteins were purified through metal affinity chromatography, and were evaluated for their humoral immunogenic properties by Western blotting with sera collected from 81 sheep naturally infected with S. Brandenburg. All 81 naturally infected sheep had IgG antibodies against recombinant FliC, V456, and V4 proteins. Furthermore, Western blotting of sera from 6 salvexinTM+B-vaccinated sheep (Trial 2004) had IgG antibodies against the 3 recombinant proteins. Whole blood cells of vaccinated sheep did not show interferon-gamma production upon stimulation with recombinant FliC and V456 proteins. Western blotting of sera from sheep vaccinated with salvexinTM and salvexinTM+B (Trial 1999), and those from rabbits vaccinated with S. Brandenburg, S. Hindmarsh and S. Typhimurium suggested that recombinant V4 contains epitopes specific for S. Brandenburg. Therefore, V4 was used to develop a novel indirect enzyme-linked immunosorbent assay (ELISA) for the detection of serum IgG antibodies in S. Brandenburg infected sheep. The ELISA showed a specificity of 100%, and a sensitivity of 93.8%. Furthermore, a new PCR assay was developed targeting rfbJ(B) gene in a single reaction, and genes invA, fliC and fljB in a multiplex reaction for the identification of S. Brandenburg from pure cultures. The sensitivity and specificity of the PCR assay was calculated to be 100%.