Antibody responses to a Cryptosporidium parvum rCP15/60 vaccine

Cryptosporidium parvum is a zoonotic apicomplexa-protozoan pathogen that causes gastroenteritis and diarrhoea in mammals worldwide. The organism is transmitted by ingestion of oocysts, which are shed in faeces, and completes its lifecycle in a single host.^1^ C. parvum is ubiquitous on dairy operations worldwide and is one of the leading causes of diarrhoea in calves on these farms.^2,3^ Here, for the first time, we describe the antibody response in a large group of cows to a recombinant C. parvum oocyst surface protein (rCP15/60) vaccine and the antibody response in calves fed rCP15/60-immune colostrum produced by these vaccinated cows. Results of recent genotype surveys indicate that calves are the only major reservoir for C. parvum infections in humans.^4^ Human C. parvum infections are particularly prevalent and often fatal in neonates in developing countries and to immunocompromised people, such as AIDs patients.^4^ Drug therapy against cryptosporidiosis is limited and not wholly efficacious in either humans or calves^5^, making development of an effective vaccine of paramount importance. To date, there is no commercially available effective vaccine against C. parvum, although passive immunization utilizing different zoite surface (glyco)proteins has showed promise.^6-9^ All cows we vaccinated produced an antibody response to the rCP15/60 vaccine and the magnitude of response correlated strongly with the subsequent level of antibody in their colostrum. All calves fed rCP15/60-immune colostrum showed a dose-dependent absorption of antibody. Our results demonstrate that vaccination of cows with rCP15/60 successfully induces antibodies against CP15/60 in their serum and colostrum and that these antibodies are then well absorbed when fed to neonatal calves. With further research, this C. parvum vaccine may well be a practical method of conferring passive protection to calves against cryptosporidiosis. Furthermore, a specifically targeted immune-colostrum may be valuable in protection and treatment of immunocompromised human patients with cryptosporidiosis

The effect of CpG-ODN on antigen presenting cells of the foal

BACKGROUND: Cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals. METHODS: Peripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNalpha, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-kappaB p65 using a chemiluminescence assay. RESULTS: The median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNalpha (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p \u3e 0.05) to adult horse cells. CONCLUSION: CpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNalpha cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment

Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions

A longitudinal prospective cohort study investigating the association of premilking stimulation and teat-end shape on milking characteristics and teat tissue condition in dairy cows

Abstract Background Premilking udder preparation is essential for harvesting high-quality milk as gently, completely, and quickly as possible. The associations between characteristics such as teat-end shape and premilking stimulation on milking characteristics and machine milking-induced changes to the teat tissue condition have not been rigorously investigated. The primary objective was to investigate the interactive effects of manual premilking stimulation (i.e., preparation lag time) and teat-end shape on total milk yield, two-minute milk yield, milking unit-on time, and time in low milk flow rate. Our secondary objective was to study the association of manual premilking stimulation and changes to the teat tissue condition after machine milking (i.e., short-term changes). In a longitudinal prospective cohort study, 384 milking observations from 129 cows were analysed. Holstein cows were housed in sand-bedded free-stall pens, fed a total mixed ration, and milked 3 times a day. Cows were classified by teat-end shape into 1 of 3 categories: pointed, flat, or round. Individual cow milking characteristics were recorded with electronic on-farm milk meters. The duration of manual stimulation, preparation lag time, and presence of short-term changes were documented for each milking observation. General linear mixed models were used to study the interactive effects of preparation lag time and teat-end shape on milking characteristics. Results There was an interaction between preparation lag time and teat-end shape for two-minute milk yield and time in low milk flow rate. The preparation lag time effect was modified by teat-end shape, while no interaction was observed for total milk yield or milking unit-on time. A generalized linear mixed model revealed that preparation lag time was associated with short-term changes in teat tissue condition, where the odds of short-term changes decreased as preparation lag time increased. Conclusions In summary, cows with different teat-end shapes may require different premilking stimulation regimens. Increasing preparation lag time benefits teat tissue condition during machine milking. Further research is warranted to optimize individual premilking stimulation in dairy cows

SAS Code for Technical note: Comparison among three methods for evaluating clinical mastitis frequency in dairy cows: cow-level risk, cow-level rate, and quarter–level rate

The lack of standardization in reporting clinical mastitis incidence limits the ability to compare results across multiple studies without additional calculations. There is both a biological and statistical rationale for evaluating the at-risk period at the quarter level. This study aimed to calculate the clinical mastitis (CM) incidence rate at quarter level in an open population prospective cohort of eight commercial dairy farms monitored from May 15, 2016 to May 31, 2017. The specific objectives were: (1) to outline an applied method for calculating CM incidence rate at the quarter level using currently available software; and (2) to compare the results of the three different measurements: cow-level incidence risk, cow-level incidence rate, and quarter-level incidence rate. All CM cases (n=7,513 milk) were identified by trained on-farm personnel, who collected all milk samples from all quarters with visibly abnormal milk. Microbiological identification was determine by culture and MALDI-TOF. All lactating quarters were at risk for CM. A quarter was at risk for a new CM case if there was at least 14 d between a previously diagnosed case and the current case in the same quarter, or if a different pathogen was isolated in the same quarter within 14 d. A total of 17,513,429 quarters days at risk (QDAR) were estimated. A SAS macros and SQL were used to bring all data together. The cow-level rate (16.6 cases per 10,000 cow-days).The quarter-level incidence rate (4.4 cases per 10,000 QDAR). The cow-level incidence risk (4.8 cases per 100 cows). Although numerically similar, it is important to note that both calculations represent different outcomes and answers different questions. Quarter-level incidence rate takes into account the sum of the time that each quarter remained under observation and at risk of developing CM. Although the evaluation of QDAR requires additional computation when compared to other methods, it might allow for a more precise evaluation of the data and a more accurate evaluation of mastitis incidence. A consensus about the methods used to report mastitis incidence will improve our ability to discuss and learn about the differences and similarities across studies, regions, and countries

The risk and control of Salmonella outbreaks in calf-raising operations: a mathematical modeling approach

Salmonellosis in calves has economic and welfare implications, and serves as a potential source of human infections. Our objectives were to assess the risk of Salmonella spread following its introduction into a herd of pre-weaned calves and to evaluate the efficacy of control strategies to prevent and control outbreaks. To meet these objectives, we developed a model of Salmonella transmission within a pre-weaned group of calves based on a well documented outbreak of salmonellosis in a calf-raising operation and other literature. Intervention scenarios were evaluated in both deterministic and stochastic versions of the model. While the basic reproduction number ($R_0$) was estimated to be 2.4, simulation analysis showed that more than 60% of the invasions failed after the introduction of a single index case. With repeated introduction of index cases, the probability of Salmonella spread was close to 1, and the tested control strategies were insufficient to prevent transmission within the group. The most effective strategies to control ongoing outbreaks were to completely close the rearing operation to incoming calves, to increase the proportion of admitted calves that were immunized ($>75$%), and to assign personnel and equipment to groups of calves

Distribution of Lactococcus spp. in New York State dairy farms and the association of somatic cell count resolution and bacteriological cure in clinical mastitis samples

We investigated the distribution of pathogenic non-agalactiae gram-positive, catalase-negative cocci (GPCN) in a convenience sample of New York State dairy farms. Our primary objective with the clinical mastitis (CM) GPCN samples was to evaluate somatic cell count (SCC) resolution and bacteriological cure of Streptococcus dysgalactiae or Streptococcus uberis versus Lactococcus lactis or Lactococcus garvieae in cows that received an approved intramammary treatment. In phase I, we assessed the distribution of the GPCN and SCC resolution. In phase II, we evaluated the SCC resolution and bacteriological cure in CM samples from the 4 farms with the highest prevalence of L. lactis or L. garvieae in phase I. In phase I, 8,868 CM and subclinical mastitis (SCM) milk samples were received from 143 farms. The GPCN samples identified by culture were confirmed with MALDI-TOF. From the 473 MALDI-TOF-confirmed GPCN samples, 155 were S. dysgalactiae (33%); 150, S. uberis (32%); 112, L. lactis (24%); 16, L. garvieae (3%); and 40, other GPCN (8%). From these, 277 were CM samples and 127 were eligible for the evaluation of SCC resolution, which was defined as SCC 64200,000 cells/mL in a composite sample 15 to 60 d post-diagnosis. The odds of SCC resolution in CM samples was evaluated with multivariable logistic regression, and the odds were 6.1 [95% confidence interval (CI):2.7\u201313.9] times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. In phase II, a total of 1,662 CM and SCM samples were evaluated with microbiological methods as in phase I, of which 211 samples were confirmed by MALDI-TOF: 39% were S. dysgalactiae (n = 61) and S. uberis (n = 21); 55%, L. lactis (n = 114) and L. garvieae (n = 2); and 6%, other GPCN (n = 13). In total, 168 CM samples were eligible for analysis and 118 were included in the final SCC resolution model. Similar statistical methods as in phase I were performed, and the odds of SCC resolution were 2.4 (95% CI: 1.1\u20135.5) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Bacteriological cure was defined as having a different or negative culture on a quarter sample taken 14 to 28 d after initial diagnosis. The odds of bacteriological cure (n = 121) were 8.0 (95% CI: 2.5\u201325.6) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Differences in SCC resolution and bacteriological cure between these groups may dictate a different management approach