In-Line Registered Milk Fat-to-Protein Ratio for the Assessment of Metabolic Status in Dairy Cows.

This study endeavors to ascertain alterations in the in-line registered milk fat-to-protein ratio as a potential indicator for evaluating the metabolic status of dairy cows. Over the study period, farm visits occurred biweekly on consistent days, during which milk composition (specifically fat and protein) was measured using a BROLIS HerdLine in-line milk analyzer (Brolis Sensor Technology, Vilnius, Lithuania). Clinical examinations were performed at the same time as the farm visits. Blood was drawn into anticoagulant-free evacuated tubes to measure the activities of GGT and AST and albumin concentrations. NEFA levels were assessed using a wet chemistry analyzer. Using the MediSense and FreeStyle Optium H systems, blood samples from the ear were used to measure the levels of BHBA and glucose in plasma. Daily blood samples were collected for BHBA concentration assessment. All samples were procured during the clinical evaluations. The cows were categorized into distinct groups: subclinical ketosis (SCK; n = 62), exhibiting elevated milk F/P ratios without concurrent clinical signs of other post-calving diseases; subclinical acidosis (SCA; n = 14), characterized by low F/P ratios (<1.2), severe diarrhea, and nondigestive food remnants in feces, while being free of other post-calving ailments; and a healthy group (H; n = 20), comprising cows with no clinical indications of illness and an average milk F/P ratio of 1.2. The milk fat-to-protein ratios were notably higher in SCK cows, averaging 1.66 (±0.29; p < 0.01), compared to SCA cows (0.93 ± 0.1; p < 0.01) and healthy cows (1.22). A 36% increase in milk fat-to-protein ratio was observed in SCK cows, while SCA cows displayed a 23.77% decrease. Significant differences emerged in AST activity, with SCA cows presenting a 26.66% elevation (p < 0.05) compared to healthy cows. Moreover, SCK cows exhibited a 40.38% higher NEFA concentration (p < 0.001). A positive correlation was identified between blood BHBA and NEFA levels (r = 0.321, p < 0.01), as well as a negative association between BHBA and glucose concentrations (r = -0.330, p < 0.01). Notably, AST displayed a robust positive correlation with GGT (r = 0.623, p < 0.01). In light of these findings, this study posits that milk fat-to-protein ratio comparisons could serve as a non-invasive indicator of metabolic health in cows. The connections between milk characteristics and blood biochemical markers of lipolysis and ketogenesis suggest that these markers can be used to check the metabolic status of dairy cows on a regular basis.

Molecular Identification of Sarcocystis Species in Sheep from Lithuania.

Data on the distribution of different Sarcocystis species in various muscles of sheep are scarce. In the present study, 190 diaphragm, oesophagus, and heart muscle samples of 69 sheep raised in Lithuania were examined for the presence of Sarcocystis spp. Under a light microscope, two morphological types of microcysts corresponding to S. arieticanis and S. tenella were detected. Eight and 12 sarcocysts of S. arieticanis and S. tenella, respectively, were isolated and characterised by the sequencing of a portion of cox1. The sequence comparisons revealed the highest similarity between European and Asian isolates of S. arieticanis and S. tenella obtained from domestic sheep and other wild Caprinae hosts. Based on peptic digestion, nested PCR targeting cox1, and sequencing, a 100% infection prevalence of S. arieticanis and S. tenella was observed in the 69 studied animals. The occurrence of S. tenella was significantly higher in the diaphragm than in the oesophagus (χ2 = 13.14, p < 0.001), whereas differences in the prevalence of S. arieticanis in the studied muscle types were insignificant (χ2 = 1.28, p > 0.05). Further molecularly based epidemiological studies are needed to compare the prevalence of Sarcocystis species in various muscles of sheep raised in different geographic regions.

Molecular identification of four Sarcocystis species in cattle from Lithuania, including S. hominis, and development of a rapid molecular detection method.

BACKGROUND: Six Sarcocystis species are known to use cattle (Bos taurus) as the intermediate host, two of which, S. hominis and S. heydorni, are zoonotic. There is a need for a method that will enable rapid identification of the Sarcocystis species in cattle. METHODS: The diaphragm muscles of 102 cattle from Lithuania were examined for the presence of Sarcocystis spp., using two different methods for species identification. Individual sarcocysts were isolated from squash preparations of the diaphragm muscle under the light microscope, followed by genetic characterisation of excised cysts using sequence analysis of the 18S rRNA (18S rRNA) and cytochrome c oxidase subunit I (cox1) genes. The same cattle muscle samples were digested and species-specific PCR analyses targeting cox1 were developed to identify the Sarcocystis isolates to the species level. RESULTS: Under the light microscope, sarcocysts were detected in 87.3% of animals, and Sarcocystis infection was verified in all digested samples. Three species, namely S. cruzi (n = 20), S. bovifelis (n = 23) and S. hirsuta (n = 6), were identified by DNA sequence analysis of isolated sarcocysts. Based on sequence analysis of cox1, the level of genetic variability depended on Sarcocystis species and geographical location. Four Sarcocystis species, S. cruzi (96.1%), S. bovifelis (71.6%), S. hirsuta (30.4%) and S. hominis (13.7%), were confirmed in the digested samples. In individual samples, the most common finding was two species of Sarcocystis (44.1%), followed by three species (26.5%), a single species (24.5%) and four species (4.9%). CONCLUSIONS: Although examination of tissue preparations under the light microscrope did not detect any sarcocysts belonging to S. hominis, this species was identified in the digested samples subjected to a cox1-specific PCR analysis. These results demonstrate the need for effective molecular diagnosis techniques to detect Sarcocystis spp., which may be present at a lower prevalence and not detectable among the limited number of sarcocysts identified individually under the light microscope.

Molecular identification of two Sarcocystis species in fallow deer (Dama dama) from Lithuania.

Due to the lack of molecular research conducted, little is known about Sarcocystis species diversity in the fallow deer (Dama dama). Until now, Sarcocystis jorrini and Sarcocystis morae were described to form sarcocysts in the muscles of this host. In the present study diaphragm muscle samples of free-ranging fallow deer from Lithuania were investigated for Sarcocystis species. Sarcocysts were detected in 39 out of 48 (81.3%) fallow deer examined. Under a light microscope two types of sarcocysts having hair-like and finger-like protrusions were observed. Based on DNA sequence analysis of cox1 and 18S rDNA, two species, S. morae and Sarcocystis entzerothi were identified. In prior studies, the latter species was only detected in Lithuanian roe deer (Capreolus capreolus) and in sika deer (Cervus nippon). The haplotype network of S. morae sequences specified close relationships between haplotypes found in the same country. According to current knowledge, the fallow deer is characterised by low Sarcocystis species richness as compared with other cervid species from Europe.