Advances in the application of genetic manipulation methods to apicomplexan parasites.

Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.

Efficacy of a control program for bovine trichomonosis based on testing and culling infected bulls in beef cattle managed under mountain pastoral systems of Northern Spain.

Bovine trichomonosis (BT) is a sexually transmitted disease that is considered a cause of early reproductive failure in cattle under extensive management conditions. Recently, Tritrichomonas foetus was detected in 41.5% of herds from one representative beef cattle breed (Asturiana de la Montaña; AM) reared in traditional mountain systems in Spain. The objective of the present study was to evaluate the effect of BT on reproductive performance and the economic consequences in AM herds. The benefits of a control program based on testing and culling infected bulls were also studied by comparing T. foetus prevalence and reproductive data before and after the implementation of the control measures. In infected herds, T. foetus infection increased calving intervals by 79 days (P<0.0001) and resulted in a higher percentage of cows-not-in calf (36% vs. 19%; P<0.001). An economic analysis showed that BT could reduce income by 68.7% in AM herds. The implementation of the control program decreased calving intervals (P<0.0001) and increased calving percentage (P<0.05). T. foetus prevalence showed a significant decline compared with the prevalence before implementing the control program (P<0.05). Nevertheless, after 2 years, the herd prevalence did not decrease (12.7-13.6%; P>0.05) and the herd incidence was 22.72%. The testing and culling policy was effective in improving reproductive efficiency but the complete elimination of BT without substantial changes in management appears unlikely because putative risk factors associated with the disease are present in the management of this breed.

Spatio-temporal epidemiology of Tritrichomonas foetus infection in Texas bulls based on state-wide diagnostic laboratory data.

Texas is the largest cattle producing state and suffers severe economic losses due to abortions caused by the protozoan parasite Tritrichomonas foetus. The objective of this study was to use data from the state-wide diagnostic laboratory system of Texas to investigate the occurrence and spatio-temporal distribution of bovine trichomoniasis (BT) in Texas, and to identify spatial disease clusters within the state. The study population consisted of bulls tested for BT in 2010 by the Texas Veterinary Medical Diagnostic Laboratory system that performs at least 95% of all T. foetus testing in the state. Preputial samples were cultured and diagnosis was made by real-time polymerase chain reaction (PCR). Data on BT was aggregated at the county level with time aggregation of one month. The scan statistics was used to identify spatial disease clusters. The database included 31,202 test results with a proportion of positives of 3.7%. As expected, BT was present throughout Texas. Testing prevalence was highest in the summer (5.5%). The scan statistics identified a spatial cluster in southeastern Texas, which could only partially be explained by cattle herd density. The findings of this study provide baseline data to monitor the success of BT control activities in Texas and aids in generating hypotheses regarding specific risk factors for the disease. The identification of high-risk areas and periods is also essential to improve intervention efforts.

Two multivalvulid myxozoans causing postmortem myoliquefaction: Kudoa megacapsula n. sp. from red barracuda (Sphyraena pinguis) and Kudoa thyrsites from splendid alfonso (Beryx splendens).

Postmortem myoliquefaction associated with multivalvulid myxozoans was found in fillets of red barracuda (Sphyraena pinguis) and splendid alfonso (Beryx splendens), which were imported to Japan from China and South Africa, respectively. Morphological examinations of the myxozoans from the somatic muscle of red barracuda revealed that spores (30.3-44.7 microm in maximum thickness) had 4 distinct winglike valves, in which 1 extremely large (12.7 x 5.8 microm), 2 small, and 1 vestigial polar capsule were present. The small subunit ribosomal DNA sequence analysis showed that the myxozoan cluster within a clade was composed of Kudoa thyrsites, Kudoa minithyrsites, and Kudoa lateolabracis, all having stellate spores with 1 polar capsule larger than the other 3. On the basis of these characteristics, we describe this parasite as Kudoa megacapsula n. sp. Morphological and molecular analyses of the myxozoan from splendid alfonso identified it as K. thyrsites, which has been described from many marine fishes. To our knowledge, this is the first record of K. thyrsites in splendid alfonso.