Joren Verbeke

Pathogen-specific incidence rate of clinical mastitis in Flemish dairy herds, severity, and association with herd hygiene

A one-year survey on clinical mastitis was conducted on 50 randomly selected commercial Flemish dairy herds to estimate the pathogen-specific incidence rate of clinical mastitis (IRCM). The severity of the cases and the potential associations with herd hygiene were studied. Participating producers sampled 845 cases and 692 dairy cows. The mean and median IRCM was estimated at 7.4 and 5.3 quarter cases per 10,000 cow-days at risk, respectively. A large between-herd variation was observed (range of 0-21.3). In general, the IRCM was lower in heifers compared with multiparous cows (2.9 vs. 11.0 quarter cases per 10,000 cow-days at risk). However, the overall IRCM in the first week after calving was higher in heifers compared with cows (43.4 vs. 31.6 quarter cases per 10,000 cow-days at risk). Streptococcus uberis (18.2% of the cases) and Escherichia coli (15.5%) were the most frequently isolated pathogens and no growth was observed in 19.9% of the cases. The majority of the cases (63.1%) were mild (only clots in milk). Moderate (hard quarter without general signs) and severe symptoms (systemic illness) were observed in 29.9 and 7.0% of the cases, respectively. Isolation of E. coli (vs. any other culture result) was more likely in moderate and severe cases compared with mild cases. Overall IRCM and E. coli IRCM were higher in dirty compared with clean herds based on udder hygiene scores (9.0 and 1.7 vs. 6.0 and 0.6 quarter cases per 10,000 cow-days at risk, respectively). This study broadens the knowledge on clinical mastitis in Flemish dairy herds and underlines the high risk of CM in early-lactation heifers, the role of the so-called environmental pathogens, and herd hygiene.

Pathogen-group specific association between CXCR1 polymorphisms and subclinical mastitis in dairy heifers

The chemokine (C-X-C motif) receptor 1 (CXCR1) gene encodes the homonymous receptor for interleukin 8 (IL8) on polymorphonuclear neutrophilic leucocytes (PMNL). Binding causes migration from blood to milk, activation and prolonged survival of PMNL, a crucial process in the innate immune defence of the bovine mammary gland against invading mastitis-causing pathogens. The main objective of this study was to screen the entire coding region of the CXCR1 gene for polymorphisms and to analyse their association with udder health of dairy heifers. One-hundred-and-forty Belgian Holstein heifers originating from 20 commercial dairy farms were genotyped by DNA sequencing. Detailed phenotypic data on udder health was available including quarter bacteriological culture results and somatic cell count (SCC) in early lactation and composite milk SCC during first lactation. In total, 16 polymorphisms (including 8 missense mutations) were detected. Polymorphism c.980A>G was associated with pathogen-group specific IMI: heifers with genotype AG were less likely to have an IMI due to major mastitis pathogens compared with heifers with genotype GG but did not have less IMI by coagulase-negative staphylococci, so-called minor pathogens. CXCR1 genotype was neither associated with quarter SCC in early lactation nor with composite SCC during lactation. Although mastitis susceptibility is influenced by many factors, some genetic polymorphisms potentially have major effects on udder health of heifers, as was shown here. These results trigger us to further study the relationship between CXCR1 polymorphisms and mastitis susceptibility in both observational and experimental trials.

Risk factors associated with subclinical mastitis as detected by California Mastitis Test in smallholder dairy farms in Jimma, Ethiopia using multilevel modelling

The prevalence of subclinical mastitis and associated risk factors at the herd, cow and quarter level were studied using multilevel modelling in smallholder dairy farms in Jimma, Ethiopia. Forty-two herds, out of the 55 dairy farms located in Jimma (76%), were visited, a questionnaire was performed, and 635 quarters belonging to 176 lactating cows were screened to detect the presence of subclinical mastitis using the California Mastitis Test (CMT). Sixty-two percent of the cows and 51% of the quarters were subclinically infected. Quarters from cows in later stage of lactation (>180 DIM) [opposed to early lactation (<90 DIM)] were more likely to be subclinically infected (OR=2.40, 95% CI=1.44-3.99). Overall, quarters from cows milked by squeezing (as opposed to stripping) were less likely to be subclinically infected (OR=0.45, 95% CI=0.29-0.71), although quarters from cows with tick-infested udders were more likely to be subclinically infected when milked by squeezing (as opposed to stripping). The milking technique did not influence the likelihood of infection in cows without ticks on the udder. This study stresses the high prevalence of subclinical mastitis in smallholder dairy farms in Jimma and a lack of awareness of the existence of the disease among dairy farmers. Implementation of a mastitis prevention program adapted to the local needs, including a focus on milking technique, application of appropriate tick control measurements as well as fertility management, allowing cows to be dried-off at a more appropriate moment, are needed. To conclude, milking by squeezing instead of stripping, but not of cows with tick-infested udders, as well as fertility management could reduce the subclinical mastitis prevalence (and incidence) on the short term.

Immune response after an experimental intramammary challenge with killed Staphylococcus aureus in cows and heifers vaccinated and not vaccinated with Startvac, a polyvalent mastitis vaccine

An experimental trial was conducted to explore the effect of vaccination with a polyvalent vaccine against mastitis (Startvac) on the early immune response after experimental intramammary challenge with a heterologous killed Staphylococcus aureus strain. The effect of vaccination on milk production, clinical signs, quarter milk somatic cell count, milk polymorphonuclear neutrophilic leukocyte (PMN) concentration and viability, the concentration of antigen-specific antibodies [slime associated antigenic complex (SAAC) and J5] and their IgG1 and IgG2 subtypes in both serum and whey, and the antigen-specific IFN-γ, IL-4, and IL-17 production by blood lymphocytes after in vitro stimulation with S. aureus and Escherichia coli extracts were determined. A cohort of 8 clinically healthy end-term cows and heifers were conveniently selected, of which half was vaccinated with Startvac at 45 and 10 d before the expected calving date and half served as nonvaccinated control animals. At 15 d in milk, 2 contralateral quarters of each of the 8 animals were challenged with 2×109 cfu/mL of the formaldehyde-killed S. aureusC195strain. The 2 other quarters were infused with phosphate-buffered saline and served as control quarters. The increase in both quarter milk somatic cell count and PMN concentration and the drop in milk production after S. aureus inoculation was less pronounced in the vaccinates than in the nonvaccinates, reflecting a less severe inflammatory response. No significant differences in PMN viability between vaccinates and nonvaccinates could be demonstrated. The serum SAAC- and J5-specific antibody concentration significantly increased across the dry period in the vaccinated animals only. The whey concentration of SAAC-specific antibodies was significantly higher in vaccinates than in nonvaccinates at both 15 and 17 d in milk, independent from the challenge status of the quarters. No significant differences in the whey J5-specific antibody concentration were observed. Vaccination with Startvac seems to primarily evoke a Th2 response for S. aureus characterized by a shift toward the IgG1 antibody subtype and accompanied by a less pronounced Th1 response. The type of response against E. coli was less clear, though a weak but significant shift toward the IgG2 antibody subtype after vaccination and high IFN-γ levels after in vitro stimulation suggest a Th1 response. The increased SAAC-specific antibody concentration in whey in vaccinates compared with nonvaccinates most probably triggers the opsonization of the inoculated S. aureus bacteria, resulting in a more efficient elimination of the bacteria from the mammary gland.

Somatic cell count and milk neutrophil viability of dairy heifers with specific CXCR1 genotypes following experimental intramammary infection with Staphylococcus chromogenes originating from milk.

Previous observational studies suggest an association between polymorphism c.980A>G in the CXCR1 gene, encoding the chemokine (C-X-C motif) receptor 1, and the innate immunity and infection status of the mammary gland. Mammary glands of eight Holstein heifers were experimentally infected with a Staphylococcus chromogenes isolate originating from a chronic intramammary infection (IMI) to study differences between CXCR1 genotypes c.980AG and c.980GG. Quarters from heifers with genotypes c.980AG and c.980GG developed subclinical mastitis but showed differences in the early response at 6-18 h post challenge. Bacterial count at 18 h post challenge tended to be higher in quarters from c.980AG heifers compared to c.980GG heifers. Somatic cell count (SCC) was higher at 6 h post challenge and tended to be higher at 9 h post challenge in c.980AG heifers compared to c.980GG heifers. Milk production decreased similarly. Milk neutrophils of c.980AG heifers showed more apoptosis at 9 h post challenge and tended to show more necrosis at 6, 9 and 12 h post challenge than c.980GG heifers. Differences were less pronounced in the later stage (>18 h) of infection. The results demonstrate that CXCR1 polymorphism can influence SCC and milk neutrophil viability following experimental IMI.

Association of CXCR1 polymorphisms with apoptosis, necrosis and concentration of milk neutrophils in early lactating dairy heifers.

Associations between polymorphisms in the candidate gene CXCR1, encoding the chemokine (C-X-C motif) receptor 1, and udder health have been identified before. In the present study, associations between the CXCR1 genotype (whole coding region) and apoptosis, necrosis, and concentration of milk polymorphonuclear neutrophilic leukocyte (PMNL) of 292 quarters belonging to 73 early lactating dairy heifers were studied. In uninfected quarters, % milk PMNL apoptosis was higher in c.980GG heifers [least squares means (LSM) 27%] compared to c.980AG heifers (LSM 16%), whereas in infected quarters, % milk PMNL apoptosis was higher in c.642GG heifers (LSM 29%) compared to c.642AG heifers (LSM 18%). Differences in milk PMNL concentration between infected and uninfected quarters were smaller in c.980AG heifers than in c.980GG heifers. An association between the CXCR1 genotype and necrosis of milk PMNL could not be demonstrated. Results indicate that CXCR1 polymorphisms influence viability and concentration of milk PMNL and provide a foundation for future research.

Pathogen group specific risk factors for clinical mastitis, intramammary infection and blind quarters at the herd, cow and quarter level in smallholder dairy farms in Jimma, Ethiopia

A cross-sectional study on clinical mastitis, intramammary infection (IMI) and blind quarters was conducted on 50 smallholder dairy farms in Jimma, Ethiopia. A questionnaire was performed, and quarters of 211 cows were sampled and bacteriologically cultured. Risk factors at the herd, cow, and quarter level for clinical mastitis and (pathogen-specific) intramammary infection were studied using multilevel modeling. As well, factors associated with quarters being blind were studied. Eleven percent of the cows and 4% of the quarters had clinical mastitis whereas 85% of the cows and 51% of the quarters were infected. Eighteen percent of the cows had one or more blind quarter(s), whereas 6% of the quarters was blind. Non-aureus staphylococci were the most frequently isolated pathogens in both clinical mastitis cases and IMI. The odds of clinical mastitis was lower in herds where heifers were purchased in the last year [odds ratio (OR) with 95% confidence interval: 0.11 (0.01-0.90)], old cows (>4 years) [OR: 0.45 (0.18-1.14)], and quarters not showing teat injury [OR: 0.23 (0.07-0.77)]. The odds of IMI caused by any pathogen was higher in herds not practicing teat drying before milking (opposed to drying teats with 1 towel per cow) [OR: 1.68 (1.05-2.69)], cows in later lactation (>180 DIM opposed to ≤90 DIM) [OR: 1.81 (1.14-2.88)], cows with a high (>3) body condition score (BCS) [OR: 1.57 (1.06-2.31)], right quarters (opposed to a left quarter position) [OR: 1.47 (1.10-1.98)], and quarters showing teat injury [OR: 2.30 (0.97-5.43)]. Quarters of cows in herds practicing bucket-fed calf feeding (opposed to suckling) had higher odds of IMI caused by Staphylococcus aureus [OR: 6.05 (1.31-27.90)]. Except for BCS, IMI caused by non-aureus staphylococci was associated with the same risk factors as IMI caused by any pathogen. No access to feed and water immediately after milking [OR: 2.41 (1.26-4.60)], higher parity [OR: 3.60 (1.20-10.82)] and tick infestation [OR: 2.42 (1.02-5.71)] were risk factors for quarters being blind. In conclusion, replacement of old cows, prevention of teat injuries/lesions, drying teats with 1 towel per cow before milking, improving fertility in order to shorten the lactation period, allowing (restricted) suckling, access to feed and water immediately after milking, and improving tick control could improve udder health in Jimma.

Milk prolactin response and quarter milk yield after experimental infection with coagulase-negative staphylococci in dairy heifers

Coagulase-negative staphylococci (CNS) are the most common bacteria involved in subclinical mastitis in dairy cows. Remarkably, CNS-infected dairy heifers produce more milk than uninfected heifers. Because the lactation hormone prolactin (PRL) is also involved in mammary gland immunity, we investigated the milk PRL response and the mammary quarter milk yield following experimental CNS challenge. Eight healthy Holstein-Friesian heifers in mid-lactation were experimentally infected using a split-udder design with 3 different CNS strains: one Staphylococcus fleurettii (from sawdust bedding) and 2 Staphylococcus chromogenes strains (one isolate from a teat apex, the other isolate from a chronic intramammary infection). Three mammary quarters per heifer were simultaneously inoculated with 1.0×10(6) cfu, whereas the remaining mammary quarter was infused with sterile phosphate-buffered saline, serving as a control. An existing radioimmunoassay was modified, validated, and used to measure PRL frozen-thawed milk at various time points until 78h after challenge. The mean milk PRL level tended to be higher in the CNS-challenged mammary quarters compared with the control mammary quarters (7.56 and 6.85ng/mL, respectively). The increase in PRL over time was significantly greater in the CNS-challenged mammary quarters than in the control mammary quarters. However, no difference was found in the PRL response when comparing each individual CNS strain with the control mammary quarters. The mean mammary quarter milk yield tended to be lower in the CNS-infected mammary quarters than in the control mammary quarters (1.73 and 1.98kg per milking, respectively). The greatest milk loss occurred in the mammary quarters challenged with the intramammary strain of S. chromogenes. Future observational studies are needed to elucidate the relation between PRL, the milk yield, and the inflammatory condition, or infection status, of the mammary gland.

Milk production, quality, and consumption in Jimma (Ethiopia): Facts and producers’, retailers’, and consumers’ perspectives

Four studies were performed to quantify milk production, quality and consumption in the town Jimma, Ethiopia. First, 47 dairy farmers and 44 milk retailers were interviewed to gain more insights in dairy farming and marketing, and associated constraints. Second, bulk milk samples (n=188) were collected for 4 consecutive weeks to investigate milk quality [Total Bacterial Counts (TBC), Coliform Counts (CC), Somatic Cell Counts (SCC), and antimicrobial residues]. Third, (bulk) milk samples from 32 farms, 46 milk retailers and the 3 local milk collection centers were collected to determine the presence of oxacillin susceptible-and oxacillin resistant Staphylococcus aureus. Fourth, 208 adult inhabitants were interviewed to gain more insight in milk consumption and associated concerns of consumers. The average dairy farm included in the studies consisted of 5 lactating cows, produced 43 liters of milk per day and was owned by male, literate adults. Milk was sold to retailers (71% of the production) and directly to customers (25%) without any quality control, whereas 4% was self-consumed. Shortage of animal nutrition and adulteration of the milk were the main constraints for farmers and retailers, respectively. The median TBC, CC and SCC were 122,500CFU/mL, 1,005CFU/mL and 609,500cells/mL, respectively. Antimicrobial residues were detected in 20% of all samples. In general, the milk quality was considered to be poor (TBC>10,000CFU/mL, and/or CC>100CFU/mL, and/or SCC>400,000cells/mL and/or presence of antimicrobial residues) in 97% of all samples. S. aureus was isolated from 12 (38%), 13 (33%), and 2 out of 3 of the milk samples originating from the dairy farms, the milk retailers, and the milk collection centers, respectively. Seven (26%) of the isolates were resistant to oxacillin suggesting the presence of MRSA (Lee, 2003). Local milk is occasionally consumed by adults but more frequently by children. Adults mainly drink spontaneously fermented milk (57% of 105 interviewees consuming local milk) whereas most milk for children is boiled (86% of 110 households with children consuming local milk). Most consumers are concerned about adulteration and milk borne diseases but not about antimicrobial residues. Educated consumers (secondary school or higher) were more likely to boil milk for own consumption, to be concerned about antimicrobial residues in milk, to be concerned about milk borne diseases and to be willing to pay more for milk with proven good quality compared to poorly educated consumers. We conclude that milk quality incentives should be introduced in Jimma, and investments should be made in knowledge transfer, training, milk collection systems and a central milk quality lab.

Polymorphism screening and association study of CXCR1 with udder health in dairy heifers

Similar to other countries, heifer mastitis is highly prevalent in Belgium. Marker assisted genetic selection against certain disadvantageous genotypes could offer possibilities to enhance host resistance. The main objective of this study was to screen CXCR1 for polymorphisms and to analyze their association with udder health during first lactation. This potential candidate gene encodes the homonymous receptor binding interleukin 8, a crucial chemokine in the innate immunity of the mammary gland. Eighty-six heifers originating from 20 Belgian farms were genotyped for the entire coding region. Fifteen single nucleotide polymorphisms (SNPs) were detected of which 10 have not been described before. Four distinct haplotypes were detected. The association between SNP at position +735 relative to the start codon (c.735C>G) and subclinical mastitis which has already been reported by other research groups could not be confirmed in our study. Instead, SNP c.642G>A was found to be associated with udder health. Prevalence of intramammary infection (IMI) in early lactation was significantly lower in heifers expressing genotype AA compared to heifers expressing genotype AG or GG. Additionally, SNP c.982A>G tended to be associated with pathogen group specific IMI. Heifers with genotype GG on this locus were more susceptible specifically to major pathogens compared to those expressing genotype AG or AA. Numerical differences in somatic cell count in early lactation and throughout lactation by CXCR1 genotype were observed but were not statistically significant. Results in this study are promising and stimulate to further elucidate the role of CXCR1 in mastitis susceptibility.