Investigation of cattle plasma proteome in response to pain and inflammation using next generation proteomics technique, SWATH-MS.

Pain assessment in farm animals has primarily relied on a combination of behavioral and physiological responses, although these are relatively subjective and difficult to quantify. It is essential to develop more effective biomarkers of pain in production animals since they are frequently exposed to routine surgical husbandry procedures. More effective biomarkers of pain would improve welfare, limit the loss of productivity associated with pain and permit better assessment of analgesics. This study aimed to investigate the use of a modern mass spectrometry data independent acquisition strategy, termed Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS), to detect candidate protein biomarkers that are known to associate with nociceptive and inflammatory processes in cattle, which could then be used to assess the efficacy of potential analgesics. Calves were randomly divided into two groups that were either surgically dehorned or subjected to restraint stress, without provision of anaesthesia or analgesia in accordance with current industry standards. Samples were analysed before and after dehorning at multiple timepoints. Significant changes in protein concentrations were detected predominantly at 24 and 96 h following dehorning, including kininogens, proteins associated with the coagulation and complement cascades and serine protease inhibitors. Gene ontology analysis revealed that the identified candidate biomarkers were associated with stress, wound healing, immune response, blood coagulation and the inflammatory and acute phase responses, which could be expected following surgical damage to tissues, but can now be more objectively assessed. These results offer more definitive and quantitative monitoring of response to tissue injury induced pain and inflammation.

Leveraging homologies for cross-species plasma proteomics in ungulates using data-independent acquisition.

The collection of blood plasma is minimally invasive, and the fluid is a rich source of proteins for biomarker studies in both humans and animals. Plasma protein analysis by mass spectrometry (MS) can be challenging, though modern data acquisition strategies, such as sequential window acquisition of all theoretical fragment ion spectra (SWATH), enable reproducible quantitation of hundreds of proteins in non-depleted plasma from humans and laboratory model animals. Although there is strong potential to enhance veterinary and translational research, SWATH-based plasma proteomics in non-laboratory animals is virtually non-existent. One limitation to date is the lack of comprehensively annotated genomes to aid protein identification. The current study established plasma peptide spectral repositories for sheep and cattle that enabled quantification of over 200 proteins in non-depleted plasma using SWATH approach. Moreover, bioinformatics pipeline was developed to leverage inter-species homologies to enhance the depth of baseline libraries and plasma protein quantification in bovids. Finally, the practical utility of using bovid libraries for SWATH data extraction in taxonomically related non-domestic ungulate species (giraffe) has been demonstrated. SIGNIFICANCE: Ability to quickly generate comprehensive spectral libraries is limiting the applicability of data-independent acquisition, such as SWATH, to study proteomes of non-laboratory animals. We describe an approach to obtain relatively shallow foundational plasma repositories from domestic ruminants and employ homology searches to increase the depth of data, which we subsequently extend to unsequenced ungulates using SWATH method. When applied to cross-species proteomics, the number of proteins quantified by our approach far exceeds what is traditionally used in plasma protein tests.

A Novel Transdermal Ketoprofen Formulation Provides Effective Analgesia to Calves Undergoing Amputation Dehorning.

There is a critical need to ensure that all cattle undergoing surgical husbandry procedures are provided effective pain relief. Non-steroidal anti-inflammatory drugs (NSAIDs) are most commonly used, and typically are administered by intramuscular (IM) injection. However, administration of NSAIDs via this route to large numbers of cattle which are handled only once or twice a year, typical of many rangeland beef production systems, presents significant occupational health and safety and mis-administration risks. To address this, a novel transdermal (TD) formulation of ketoprofen was developed, and its efficacy assessed in a study of 36 Holstein-Friesian calves which were assigned to a placebo (n = 10), a TD ketoprofen (n = 10), an IM ketoprofen (n = 10) and sham dehorned group (n = 6). TD ketoprofen significantly reduced plasma cortisol concentrations between 1 to 4 h after dehorning compared to placebo treated calves, with concentrations at 2 and 4 h being very similar to those for sham dehorned calves. The expected log count of positively associated pain variables (ear flick, tail wag, ruminating, head shake, lying down, grooming and neck extending) in the TD group was reduced by 42%, compared to placebo calves, with an overall significant (p < 0.05) treatment effect. The IM group exhibited similar responses and both TD and IM cattle had a higher BW gain at 2 and 5 (p < 0.05) weeks post-dehorning, compared to placebo. This study has shown that TD administered ketoprofen was at least as effective as IM to control pain associated with dehorning and facilitates the administration of analgesic drugs prior to the surgical husbandry procedures being performed.

Patho-epidemiological study on Genotype-XIII Newcastle disease virus infection in commercial vaccinated layer farms.

AIM: The present research work was carried out to study the patho-epidemiological aspects of Genotype-XIII Newcastle disease virus (NDV) infection in commercial layer in and around Anand, Gujarat. As the outbreaks have reported in vaccinated flocks, it was felt necessary to study the disease with respect to its changing pathogenicity and relevant aspects. MATERIALS AND METHODS: The study comprised of patho-epidemiology of Newcastle disease (ND) by information collected from different layer farms suffering from the disease in relation to incidence pattern and mortality, duration of mortality, susceptible age, and loss due to production performance. Clinical signs were recorded based on observations. During post-mortem, gross lesions were also recorded. For histopathological examination visceral organs according to lesions were collected in 10% formalin and processed slide stained by hematoxylin and eosin for microscopic examination. Cultivation of virus was done in embryonated specific pathogen-free (SPF) eggs of 9-11 days and isolation of virus was done for haemagglutination (HA) and haemagglutination inhibition (HI) test and to identify pathotype of virus by intracerebral pathogenicity index (ICPI) test to determine the virulence of virus. The Genotype-XIII NDV was confirmed by F gene sequence and whole genome sequence. RESULTS: During the study mortality due to ND was recorded in 13 layer flocks in spite of routine vaccination, which usually contain Genotype-II strain of virus. The mortality was observed as high as above 50% with an average of 21.21%. The susceptible age for disease was found to be 6-14 weeks. The duration of mortality observed was 23 days. The disease resulted in a significant reduction in body weight, feed intake and drop in egg production. Majority of the outbreaks appeared during extremely hot months of April to June. Greenish diarrhoea was frequently seen in birds that survived early in infection. Mortality continued for 2-3 weeks and reduced with appearance of torticollis. Gross lesions were characterized by multifocal to diffuse hemorrhages around proventricular glands, necrotic (diphtheritic) haemorrhagic ulcers throughout the intestine, disseminated multiple foci of necrosis and pin-point hemorrhages in the spleen parenchyma. The microscopic lesions include focal to diffuse hemorrhages, diffuse infiltration of mononuclear cells, necrosis, and degeneration in visceral organs. All the 13 farm samples (n=13) resulted in death of all the embryos following incubation up to 72 h post-inoculation. All the 13 allantois fluids from field samples along with F and R2B vaccine sample were found positive for HA activity, which was further confirmed by HI using known NDV serum. The values of ICPI were 2.0 which were indicative of velogenic nature of the field NDV strain. CONCLUSION: The study indicated that presently available live and attenuated vaccines which include Genotype-II NDV have failed in protecting the flocks against Genotype-XIII and resulted in outbreaks with mortality above 50%. ICPI score of 2.0 confirmed that the present outbreaks were due to Genotype-XIII NDV, which is velogenic in nature.