Subclinical mastitis in dairy cows in major milk-producing areas of Sri Lanka: Prevalence, associated risk factors, and effects on reproduction.

Compared with clinical mastitis, the subclinical form of mastitis (SCM) is more common and thought to cause more economic losses to the dairy industry. The current study aimed to investigate the prevalence, risk factors of SCM, and effects on reproduction of dairy cows in major milk-producing areas of Sri Lanka. A total of 1,357 cows of selected farms in 3 regions were examined in the study. California Mastitis Test was conducted for individual cows, and a score of 2 or more for any quarter without any clinical symptoms and abnormalities in milk was considered as positive for SCM. Samples from infected animals were collected and subjected to bacteriological analysis. A pretested questionnaire was used to collect data on individual cows and herds. Risk factors associated with SCM were analyzed using binary logistic regression in generalized linear mixed models. The effect of SCM on calving to conception interval and days from calving to artificial insemination were analyzed by survival analysis using Cox's proportional hazards regression and Kaplan-Meier survival function estimates, respectively. A Poisson regression model was run to determine the effect of SCM on number of artificial inseminations per conception. The prevalence of SCM was 57.5, 11.8, and 45.5% in the regions A, B, and C, respectively. The most common pathogen was Staphylococcus aureus, with 87.1, 56.5, and 92.3% in the regions A, B, and C, respectively. Logistic regression analysis showed that parity, farming system, milking area, region, and herd significantly affect the prevalence of SCM. Subclinical mastitis during the pre-breeding period was associated with 14% increase in the chance of having a greater number of artificial inseminations per conception. Likewise, median days from calving to artificial insemination was longer in cows with SCM compared with normal cows (79 and 64 d, respectively). Therefore, SCM affected the inseminated proportion of studied cows. However, SCM was not associated with the calving to conception interval. The results revealed that the cow factors and milk hygiene play a significant role in the prevalence of SCM.

Distribution of infectious endogenous retroviruses in mixed-breed and purebred cats.

Endogenous retroviruses of domestic cats (ERV-DCs) are members of the genus Gammaretrovirus that infect domestic cats (Felis silvestris catus). Uniquely, domestic cats harbor replication-competent proviruses such as ERV-DC10 (ERV-DC18) and ERV-DC14 (xenotropic and nonecotropic viruses, respectively). The purpose of this study was to assess invasion by two distinct infectious ERV-DCs, ERV-DC10 and ERV-DC14, in domestic cats. Of a total sample of 1646 cats, 568 animals (34.5%) were positive for ERV-DC10 (heterozygous: 377; homozygous: 191), 68 animals (4.1%) were positive for ERV-DC14 (heterozygous: 67; homozygous: 1), and 10 animals (0.6%) were positive for both ERV-DC10 and ERV-DC14. ERV-DC10 and ERV-DC14 were detected in domestic cats in Japan as well as in Tanzania, Sri Lanka, Vietnam, South Korea and Spain. Breeding cats, including Singapura, Norwegian Forest and Ragdoll cats, showed high frequencies of ERV-DC10 (60-100%). By contrast, ERV-DC14 was detected at low frequency in breeding cats. Our results suggest that ERV-DC10 is widely distributed while ERV-DC14 is maintained in a minor population of cats. Thus, ERV-DC10 and ERV-DC14 have invaded cat populations independently.

Influence of bovine sub-clinical mastitis and associated risk factors on calving interval in a population of crossbred lactating cows in Sri Lanka.

A study was carried out to identify prevalence and risk factors for bovine subclinical mastitis (SCM) in crossbred lactating cows within the Kurunagala district of Sri Lanka. In this study, 283 crossbred cows (Jersey x Shahiwal) from randomly selected medium- and relatively large-scale dairy farms were screened for SCM. The Californian mastitis test (CMT) plus microbiological analyses were employed for screening. A pre-tested questionnaire was used to collect information on the cows and their management. When screening was based only on CMT, the prevalence of SCM was 49%, and it was 44% if based on both CMT and pathogen isolation. The isolated pathogens were Staphylococcus spp. (80%, 98/123), Escherichia coli (14%, 17/123), Streptococcus spp. (3%, 4/123) and Pasteurella multocida (3%, 4/123). The age, parity and milk yield showed positive correlations with SCM (p < 0.05). The prevalence of SCM was relatively high after the fifth parity (p = 0.03, odds ratio = 9.49). A positive correlation of increase in calving interval (more than 18 months) with SCM was observed. The prevalence of SCM on the dairy farms can be reduced by implementing more hygienic management practices.

Identification of essential residues of CTLA-2alpha for inhibitory potency.

To identify functionally essential sequences and residues of CTLA-2alpha, in vitro mutagenesis was carried out. The coefficient of inhibition (K(i)) was determined towards rabbit cathepsin L using Z-Phe-Arg-MCA as the substrate. Recombinant CTLA-2alpha inhibited the enzyme potently (K(i) = 15 nM). A truncated mutant, lacking the N- and C-terminal Ala1-Asp9 and Leu80-Glu109 regions, was also a potent inhibitor (K(i) = 10 nM). Subsequent short deletions in the central region (Asn10-Ser79) showed three functionally essential distinct regions: Asn10-Phe19, His30-Ala44 and Ser55-Ser79. These regions cover sequences corresponding to three helices (alpha1, alpha2 and alpha3) and sequences that interact with the cognate enzyme. Alanine scanning showed that replacement of one of three conserved Trp residues increased the K(i) by 15-20-fold; whereas, replacement of two/three Trp residues at once caused complete loss of potency, as did replacing Cys75 with Ala or Ser. The proteins from wild-type (WT) CTLA-2alpha and mutant C75A were stable overnight when incubated with cathepsin L; whereas, proteins from mutants W12A, W15A and W35A were quickly digested. Incubation of cathepsin L/WT CTLA-2alpha formed a complex; whereas, C75S did not form a complex. Our overall results point to a critical role of W12, W15, W35 and Cys75 residues in CTLA-2alpha.

Dendritic and axonal localization of cytotoxic T-lymphocyte antigen-2 alpha protein in mouse brain.

Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2alpha) is a novel cysteine proteinase inhibitor protein originally discovered and expressed in mouse activated T-cells and mast cells. Expressed recombinant CTLA-2alpha is shown to exhibit selective inhibition of cathepsin L-like cysteine proteinases. We have recently reported the expression pattern of CTLA-2alpha mRNA in mouse brain by in situ hybridization, demonstrating that it is mainly enriched within neuronal populations. In this study we present the distribution profile of the protein by immunohistochemical analysis. Results showed that CTLA-2alpha protein is preferentially localized in dendritic and axonal compartments. In telencephalon, strong labeling was detected in dendrites in the cerebral cortices, stratum radiatum and stratum lacunosum moleculare and within axonal fibers of stratum lucidum where mossy fibers emanating from all parts of the granule cell layer of dentate gyrus terminate at pyramidal neurons and interneurons. In diencephalon, moderate staining was found in all thalamic nuclei but was strong in medial habenular nucleus and the hypothalamic nuclei including suprachiasmatic nucleus, optic chiasm, arcuate nucleus and median eminence. In mesencephalon, strong immunoreactivity was detected in superior colliculus, inferior colliculus and paramedian raphe nucleus. In the rhombencephalon, the pontine nucleus and transverse fibers of the pons revealed strong staining but were moderate in vestibular nuclei. Strong immunoreactivity was also observed in the internal white matter, granule cell layer and Purkinje cell layer within cerebellum. On Western blot analysis, a band of 14 kDa for CTLA-2alpha from protein extracts of the cerebrum, cerebellum, pons and medulla was detected. The distribution pattern and functional considerations of CTLA-2alpha in the brain are discussed.

Expression mapping of cytotoxic T-lymphocyte antigen-2alpha gene transcripts in mouse brain.

Cytotoxic T-lymphocyte antigen-2alpha (CTLA-2alpha), an inhibitor peptide homologous to the proregion of mouse cathepsin L, was originally discovered and expressed in mouse-activated T-cells and mast cells. Expressed recombinant CTLA-2alpha is shown to exhibit selective inhibition to cathepsin L-like cysteine proteinases. However, its in vivo targets in mammalian tissues are yet to be identified. We carried out in situ hybridization studies to examine the expression pattern of CTLA-2alpha mRNA and determine the specific cell types synthesizing CTLA-2alpha in the mouse brain. CTLA-2alpha mRNA was detected in various neuronal populations within the telencephalon in cerebral cortices, olfactory system, septum, basal ganglia, amygdala and highest levels were observed in the hippocampus. Within the diencephalon high density of positive cells was found in mediodorsal and lateral posterior thalamic nuclei and medial habenular nucleus (MHb). In the hypothalamus, high density of CTLA-2alpha mRNA labeling was seen in the suprachiasmatic nucleus (Sch), optic tract, arcuate nucleus, and median eminence. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus were also densely labeled. Other mesencephalic expression sites were the superior colliculus, periaqueductal gray, paramedian raphe nucleus, and inferior colliculus. In the rhombencephalon, strong labeling was detected in the pontine, vestibular, and reticular nuclei. Intense expression was also noted within cerebellar cortex in Purkinje neurons and at a moderate level in granule cell layer, stellate, and basket cells. A possible function of this novel inhibitor peptide in relation to learning, memory, and diseases is discussed.