The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows

Twelve 2-year old heifers in their fifth month of gestation when pregnancy tested were used in this study. Six heifers aborted at approximately 4 months of gestation and had blood samples drawn less than 6 weeks after the abortions were identified. Blood samples were also drawn from three sero-positive pregnant and three sero-negative pregnant heifers. DNA was isolated from the samples and a 350 bp fragment of the Nc-5 gene was PCR amplified using primer pair Np21+ and Np6+. Also, the Internal Transcribed Spacer 1 (ITS1) was PCR amplified using Tim 3 and Tim 11 primer pair. The Nc-5 gene fragment was cloned, sequenced and the sequence BLAST-tested. Similarly, the ITS1 product was sequenced and BLAST-tested. The BLAST test results revealed that Neospora caninum DNA was present in these blood samples indicating that polymerase chain reaction can be used in the detection of N. caninum DNA in the blood of sero-positive cows.     

Validation of the FAMACHA eye color chart for detecting clinical anemia in sheep and goats on farms in the southern United States

Recent studies on sheep and goat farms in the southern United States indicate that multiple-anthelmintic resistance in Haemonchus contortus is becoming a severe problem. Though many factors are involved in the evolution of resistance, the proportion of the parasite population under drug selection is believed to be the single most important factor influencing how rapidly resistance develops. Therefore, where prevention of resistance is an important parallel goal of worm control, it is recommended to leave a portion of the animals untreated. Recently, a novel system called FAMACHA was developed in South Africa, which enables clinical identification of anemic sheep and goats. When H. contortus is the primary parasitic pathogen, this system can be applied on the farm level to reduce the number of treatments administered, thereby increasing the proportion of the worm population in refugia. Since most studies validating the FAMACHA method have been performed in South Africa, it is important that the method be tested in other regions before its use is broadly recommended. We performed a validation study of FAMACHA by testing the system in sheep (n = 847) and goats (n = 537) of various breeds and ages from 39 farms located in Arkansas, Georgia, Louisiana, Florida, and the US Virgin Islands. The color of the ocular conjunctiva of all animals were scored on a 1-5 scale using the FAMACHA card, and blood samples were collected from each animal for determination of packed cell volume (PCV). Fecal samples were also collected from a majority of the animals tested for performance of fecal egg counts (FEC). Correlations between PCV and eye scores, PCV and FEC, and FEC and eye scores were all highly significant for both sheep and goats (P < 0.001). Data for both FAMACHA scores and PCV were evaluated using two separate criteria for anemia: eye score values of 3, 4 and 5 or 4 and 5, and PCV values of < or =19 or < or =15 were considered anemic. Specificity was maximized when eye score values of 4 and 5 were considered anemic and PCV cut off for anemia was < or =19, but sensitivity was low. In contrast, sensitivity was 100% for both sheep and goats when eye score values of 3, 4 and 5 were considered anemic and PCV cut off was < or =15, but specificity was low. In both sheep and goats, predictive value of a negative was greater than 92% for all anemia and eye score categories, and was greater than 99% for both eye score categories when an anemia cutoff of < or =15 was used. Predictive value of a positive test was low under all criteria indicating that many non-anemic animals would be treated using this system. However, compared to conventional dosing practices where all animals are treated, a large proportion of animals would still be left untreated. These data indicate that the FAMACHA method is an extremely useful tool for identifying anemic sheep and goats in the southern US and US Virgin Islands. However, further studies are required to determine optimal strategies for incorporating FAMACHA-based selective treatment protocols into integrated nematode control programs.     

Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates. METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent antibody test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice. RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3-7%. CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

Virulence genes of Escherichia coli strains isolated from mastitic milk

Escherichia coli, a Gram-negative environmental pathogen associated with bovine mastitis was isolated from the milk of 34 symptomatic cows that had been diagnosed with clinical mastitis. Eighty isolates were obtained over a 17-month period and these isolates were screened by DNA amplification for the following E. coli virulence genes: cnf1, cnf2, eaeA, eagg, einv, ltx1, stx1, stx2 and vt2e. Thirty of the bacterial isolates, obtained from 23 different cows, had toxin genes identified in their DNA. The most common virulence gene detected was stx1, with a prevalence of 31%, followed by cnf2 (7.5%), vt2e (6.25%) and eaeA (4%). The possession of different virulence genes by the bacterial isolates had no discernable impact on the health status of the cows as there was no correlation between the potential for toxin production by the E. coli isolates and the systemic clinical condition of the respective infected cows.     

Comparison of a camera - software system and typical farm management for detecting oestrus in dairy cattle at pasture

AIM: To compare the sensitivity, specificity, predictive values and accuracy of detection of oestrus using a novel oestrus detection strip (ODS) and a camera-software device (CSD) with typical farm management practices of visual observation and use of tail paint in dairy cattle at pasture. METHODS: Dairy cows (n = 480) in a seasonal-calving herd managed at pasture under typical commercial conditions in New Zealand were stratified by age, body condition score and days in milk, then randomly allocated to one of two groups prior to the planned start of mating (PSM). Tail paint was applied to all cows and oestrus detected by visual observation of oestrous behaviour and removal of paint, by farm staff. One group (n = 240) was fitted with ODS and also monitored for signs of oestrus using a CSD, while the Control group (n = 240) was monitored using tail paint and visual observations only. Cows detected in oestrus were artificially inseminated (AI), and pregnancy status determined using rectal palpation and ultrasonography, 51-52 days after the end of a 55-day A period. Results of pregnancy diagnosis were used to confirm the occurrence of oestrus, and the sensitivity, specificity, predictive value and accuracy of detection of oestrus compared between oestrus detection methods. RESULTS: The sensitivity and accuracy of oestrus detection in the Control group, using visual observation and tail paint, were low. Compared with the Control group, detection of oestrus using the ODS and CSD resulted in greater sensitivity (85% vs 78%; p = 0.006), specificity (99.6% vs 98.0%; p < 0.001), positive predictive value (PPV; 88% vs 51%; p < 0.001) and overall accuracy (99.0% vs 98.0%; p < 0.001). Negative predictive value (NPV) did not differ significantly between groups (99.4% vs 99.3%; p = 0.28). Pregnancy rate to first service was higher in the CSD group than in the Control group (72% vs 39%; p < 0.05). Use of the CSD significantly increased the cumulative proportion of cows pregnant to AI over the breeding period (p = 0.044). CONCLUSION AND CLINICAL RELEVANCE: The ODS and CSD was satisfactory for detection of oestrus in seasonal calving dairy herds grazing on pasture and could improve the sensitivity and accuracy of detection of oestrus in herds where these are low.     

Mutations in the sodium channel associated with pyrethroid resistance in the greenhouse whitefly, Trialeurodes vaporariorum

BACKGROUND: Trialeurodes vaporariorum Westwood is an important pest of protected crops in temperate regions of the world. Resistance to pyrethroid insecticides is long established in this species, but the molecular basis of the mechanism(s) responsible has not previously been disclosed. RESULTS: Mortality rates of three European strains of T. vaporariorum to the pyrethroid bifenthrin were calculated, and each possessed significant resistance (up to 662‐fold) when compared with a susceptible reference strain. Direct sequencing revealed three amino acid substitutions in the para‐type voltage‐gated sodium channel (the pyrethroid and DDT target site) of bifenthrin‐resistant T. vaporariorum at positions previously implicated with pyrethroid or DDT resistance (M918L, L925I and T929I) in other related species. CONCLUSION: This study indicates that resistance to bifenthrin in T. vaporariorum is associated with target‐site insensitivity, and that the specific mutations in the sodium channel causing resistance may differ between localities. Copyright © 2012 Society of Chemical Industry     

Experimental comparison of aerial larvicides and habitat modification for controlling disease-carrying Aedes vigilax mosquitoes

BACKGROUND: Microbial and insect‐growth‐regulator larvicides dominate current vector control programmes because they reduce larval abundance and are relatively environmentally benign. However, their short persistence makes them expensive, and environmental manipulation of larval habitat might be an alternative control measure. Aedes vigilax is a major vector species in northern Australia. A field experiment was implemented in Darwin, Australia, to test the hypotheses that (1) aerial microbial larvicide application effectively decreases Ae. vigilax larval presence, and therefore adult emergence, and (2) environmental manipulation is an effective alternative control measure. Generalised linear and mixed‐effects modelling and information‐theoretic comparisons were used to test these hypotheses. RESULTS: It is shown that the current aerial larvicide application campaign is effective at suppressing the emergence of Ae. vigilax, whereas vegetation removal is not as effective in this context. In addition, the results indicate that current larval sampling procedures are inadequate for quantifying larval abundance or adult emergence. CONCLUSIONS: This field‐based comparison has shown that the existing larviciding campaign is more effective than a simple environmental management strategy for mosquito control. It has also identified an important knowledge gap in the use of larval sampling to evaluate the effectiveness of vector control strategies. Copyright © 2011 Society of Chemical Industry     

A survey of feeding,management and faecal pH of Thoroughbred racehorses in the North Island of New Zealand

AIM: To identify feeding and management variables associated with variation in faecal pH within a population of intensively managed Thoroughbred racehorses in New Zealand.

METHODS: A cross-sectional survey was conducted of 16 racehorse trainers in the North Island of New Zealand. Interviews were conducted at the trainers' stables to obtain information on feeding and management of horses, and faecal samples were collected and faecal pH measured.

RESULTS: Ninety-seven percent of the horses surveyed were confined in an area ≤5 × 5 m for ≥12 h/day. Trainer's age, number of years they had trained horses, age and gender of horses, weeks in race training, racing class, frequency of feeding, bedding type, and exercise workload had no effect on mean faecal pH. Acidic faecal pH (pH ≤6.32) was associated with stables with ≤12 horses, and trainers at stables with ≤12 horses offered more concentrate feed than those at stables with >12 horses. Acidic faecal pH was associated with trainers who offered 4 kg of grain as the only form of concentrate fed, or offered ≤2.25 kg hay/day. Horses that displayed stable vices had less acidic faecal pH than horses that did not display stable vices, viz pH 6.70 (standard error of the mean (SEM) 0.135) vs 6.43 (SEM 0.029) (p=0.04).

CONCLUSIONS AND CLINICAL RELEVANCE: Racehorse management in New Zealand is similar to that observed in other major racing countries. Trainers with ≤12 horses fed more concentrates and their horses had lower faecal pH than those of trainers with >12 horses. Irrespective of management system, it appears important to provide at least 2.25 kg of hay/day ad libitum, to buffer hindgut acidosis associated with diets high in soluble carbohydrate.     

Preliminary observation on a new chemotherapeutic, “Enterol”, and its possible uses in the treatment of salmonellosis

Extract

“Enterol” (I.C.I.) was first used in a moribund pig with rather astonishing results. From the symptoms described, it was presumed that it had salmonellosis. As a result of this observation, the writer was asked to try the drug on known salmonellosis cases.