Comparison of duplex PCR and microscopic techniques for the identification of Babesia bigemina and Babesia bovis in engorged female ticks of Boophilus microplus

A single-step duplex polymerase chain reaction (PCR) technique and traditional microscopic examination of haemolymph smears were used to detect Babesia bigemina and/or Babesia bovis infection in engorged female ticks of Boophilus microplus recovered from calves raised in an endemic area of the State of Minas Gerais, Brazil. In the PCR amplification of tick-derived DNA, pairs of oligonucleotide primers specific for a 278-bp sequence from B. bigemina and for a 350-bp sequence from B. bovis were used conjointly. The microscopic examination of haemolymph revealed that 16.7% of the engorged ticks were infected with Babesia spp., although no significant differences (rho > 0.05) were found in the infection rate of ticks collected from calves of different age groups. PCR analysis showed that 77.8% of the engorged ticks whose haemolymph contained sporokinetes were infected with B. bigemina, 7.8% with B. bovis and 14.4% with both protozoan species. However, the PCR assay further revealed that, amongst the engorged female ticks whose haemolymph was apparently negative for the presence of sporokinetes, 15.6% were infected with B. bigemina, 2.2% with B. bovis and 10.0% with both species. The duplex PCR method is thus more efficient and sensitive than the microscopic assay and also permits facile identification of the protozoa species present in engorged female ticks.     

A 3-year assessment of nitrous oxide emission factors for urine and dung of grazing sheep in a subtropical ecosystem

Purpose

Grazing livestock has strong impact on global nitrous oxide (N₂O) emissions by providing N sources through excreta. The scarcity of information on factors influencing N₂O emissions from sheep excreta in subtropical ecosystems such as those of Southern Brazil led us to conduct field trials in three different winter pasture seasons on an integrated crop–livestock system (ICL) in order to assess N₂O emission factors (EF-N₂O) in response to variable rates of urine and dung.

Materials and methods

The equivalent urine-N loading rates for the three winter seasons (2009, 2010, and 2013) ranged from 96 to 478 kg ha^?1, and the dung-N rates applied in 2009 and 2010 were 81 and 76 kg ha^?1, respectively. Air was sampled from closed static chambers (0.20 m in diameter) for approximately 40 days after excreta application and analyzed for N₂O by gas chromatography.

Results and discussion

Soil N₂O-N fluxes spanned the ranges 4 to 353 μg m^?2 h^?1 in 2009, ??47 to 976 μg m^?2 h^?1 in 2010, and 46 to 339 μg m^?2 h^?1 in 2013. Urine addition resulted in N₂O-N peaks within for up to 20–30 days after application in the 3 years, and the strength of the peaks was linearly related to the N rate used. Emission factors of N₂O (EF-N₂O, % of N applied that is emitted as N₂O) of urine ranged from 0.06 to 0.34% and were essentially independent of N rate applied. By considering a ratio of N excreted by urine and dung of 60:40, a single combined excretal EF-N₂O of 0.14% was estimated.

Conclusions

Our findings showed higher mean EF-N₂O for sheep urine than that for dung (0.21% vs 0.03%), irrespective of the occurrence or not of urine patches overlap. This value is much lower than default value of 1% of IPCC’s Tier 1 and reinforces the needs of its revision.

     

Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.     

Camel trypanosomosis in the Canary Islands: assessment of seroprevalence and infection rates using the card agglutination test (CATT/T. evansi) and parasite detection tests

Trypanosomosis due to Trypanosoma evansi (surra) is a major enzootic disease of the dromedary camel. Thus, the purpose of the present study was to assess seroprevalence and infection rates in the Canary Islands using antibody(-card agglutination test-CATT/T. evansi) and parasite detection tests (micro-Haematocrit Centrifugation technique, Giemsa stained blood smears, microscopic examination of lymph node aspirates and mouse inoculation). PCV was also determined. 745 dromedary camels (483 females and 262 males) were examined. Trypanosomes were detected in seven animals. 36 animals yielded CATT positive results while 709 animals were negative. All parasitologically positive animals were also CATT positive. Results showed a good correlation between CATT positive and low PCV and a higher seroprevalence in older animals. Trypanocidal drugs have not been registered in Spain and, consequently, if vigilance is not exercised the prevalence could be increased in the future.