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Abstract

The immune response of cows against the core oligosaccharide of Escherichia coli rough mutants (core types R1-R4, K-12 and J-5) was investigated after immunization with a synthetic glycoconjugate composed of deacylated LPS conjugated to hemocyanine (22 animals). Ab formation was measured by ELISA using LPS or deacylated LPS conjugated to BSA as an Ag. The glycoconjugate immunogens were used to vaccinate cows (36 animals), which were then challenged intramammarily with E. coli O 157 (K1 negative, R1 core type). Compared with control groups no protection was observed, although high titers against the R1 core type were detected in vaccinated animals. Western blots using the immune sera showed that the Ab response was directed against the core region and not against the O-antigen, which may explain the failure of the vaccine.

Keywords

Immune response endotoxin lipopolysaccharide serum Abs vaccine bovine mastitis

Introduction

Bovine mastitis is a frequently occurring infection of lactating cows, which is an important economical factor in milk industry.^1–9 Staphylococcus aureus and Escherichia coli are the major pathogens causing mastitis in cows and several attempts have been made to develop vaccines against this disease whereby a vaccine using killed E. coli J-5 bacteria is currently used in North America with differing success.^10–19 The J-5 strain is a rough mutant, which has a defect in LPS biosynthesis and does not contain in its LPS the O-side chain and the outer core.²⁰ This strain has been claimed, since the late 1960s, to induce Abs in mice, rabbits and humans that have a broad serological cross-reactivity and a broad cross-neutralizing activity against LPS of Gram-negative bacteria in general.²¹ Even various mAbs have been obtained after immunization with J-5 LPS with unequivocal results in protection experiments (reviewed in Müller-Loennies, et al.²²).

In the 1990s, a mAb (WN1 222-5) was developed that was able to neutralize the endotoxin of all E. coli strains of all O-serotypes, and was considered useful for the treatment of human Gram-negative septicemia.²³ However, this Ab was not introduced into clinical use as a major percentage of human Gram-negative sepsis is caused by Klebsiella pneumoniae and Pseudomonas aeruginosa, in addition to E. coli.

We therefore hypothesized that the induction of Abs similar to WN1 222-5 could be the basis for the development of a new vaccine against bovine mastitis.

Materials and methods

Bacterial LPS and neoglycoconjugates

LPS from rough mutants of E. coli Rc (strain J-5), R1 (strain F470), R2 (strain F576), R3 (strain F653), R4 (strain F2513) and K-12 (strain 3110) were extracted with phenol/chloroform/light petroleum ether as described previously.²⁴ LPS was deacylated (LPS_deac) and conjugated to BSA as reported elsewhere; it will be abbreviated as LPS_deac-BSA.²⁵ For the preparation of neoglycoconjugates used for immunization, oligosaccharides were conjugated to hemocyanine (HC) following the same protocol; these conjugates will be abbreviated as LPS_deac-HC.

Immunization with LPS_deac-HC glycoconjugates

In the first experiment, 3 cows (conventional Dutch dairy cows, 10–20 months of age before first immunization) were each immunized with LPS_deac-HC containing the LPS_deac of E. coli R1, R2, R3, R4, K-12 or J-5 and alu-oil as adjuvant (alu-oil is a water-in-oil emulsion containing aluminium hydroxid). The animals received 2 ml of vaccine intramuscularly containing 250 µg of conjugate per dose and alu-oil as adjuvant on d 0, 42 and 126. Blood samples were taken at d 0, 28, 42, 56, 84, 126 and 140 by puncture of the tail vein using vacutainers; sera were stored at −20℃.

In a second experiment, 4 cows (conventional Dutch dairy cows, 10–20 months of age before first immunization) were immunized with a mixture of LPS_deac-HC containing the LPS_deac of E. coli R1, R2, R3, R4 and K-12. The animals were vaccinated intramuscularly with 3 ml of vaccine containing 150 µg of each per dose and alu-oil as adjuvant on d 0 and 42. Blood samples were taken at d 0, 28, 42 and 56 by puncture of the tail vein using vacutainers and sera were stored at −20℃.

Vaccination and challenge

In the final experiment, we immunized cows (conventional Dutch dairy cows; first parity, in lactation for 2–3 months) in groups of 12 animals with LPS_deac-HC glycoconjugate of E. coli R1 (150 µg per dose in 2 ml) or a mixture of R1, R2 and R3 (150 µg of each per dose in 2 ml) on d 0, 42 and 84 intramuscularly; both vaccines contained alu-oil as adjuvant. A control group of 12 animals was injected with the adjuvant alone. Two wk later the animals were challenged intramammarily with an E. coli wild type strain (O-157, K1 negative, R1 core type) isolated from a mastitis case. Sera were collected over a period of 119 days and tested by LPS-ELISA or by conjugate-ELISA using LPS or LPS_deac of E. coli R1, R2 or R3 as antigen.

Clinical measurements

For clinical evaluation, rectal temperatures were measured twice daily from 1 d before challenge, up to 3 d after challenge or until rectal temperatures were <39.0℃. To determine if vaccination prevented or reduced intramammary infection after challenge with E. coli, somatic cell counts of quarter foremilk and the number of CFU of E. coli in fore milk samples were determined regularly from the day before and up to 21 d after challenge.

Core typing of clinical bovine mastitis isolates

In 50 clinical isolates of E. coli mastitis strains the core type of LPS was determined. Bacterial strains were grown on solid media overnight, a loopful of bacteria was suspended in lysing buffer as used for SDS-PAGE and digested with proteinase K. Aliquots (1–5 µl) were dotted onto nitrocellulose. After drying (1 h, 37℃) and fixation (5 min, room temperature, 10% isopropyl alcohol, 10% acetic acid), the nitrocellulose was washed twice shortly (immediate removal of the washing fluid) and twice (5 min each) with distilled water. All the following steps were performed at room temperature. After blocking in blotting buffer (50 mM Tris-HCl, 0.2 M NaCl, pH 7.4) supplemented with 10% non-fat dry milk for 1 h, core-specific mAbs were added. After incubation overnight and washing (6 times, 5 min each) in blotting buffer, alkaline phosphatase-conjugated goat-anti mouse IgG (heavy and light chain specific; Dianova) was added (diluted 1:1,000 in blotting buffer supplemented with non-fat dry milk) and incubation was continued for another 2 h. After washing as before, 5-bromo-4-chloro-3-indoylphosphate and p-toluidine p-nitroblue tetrazolium chloride (Bio-Rad, Munich, Germany) were added as substrates according to the manufacturer’s instructions. After 15 min the reaction was stopped by the addition of distilled water.

The following mAb were used for the development: mAb X1SW1, mAb S37-20 and mAb S31-14, being specific for the R1, R3 and K-12 core type respectively. MAb H7 41-76 recognized the R2 and K-12 core type; therefore, strains being positive with this mAb, but negative with the K-12 specific Ab S31-14, were classified as R2 core type. Those which did not react with any of the Abs were classified as R4 core type.

Serology

ELISA using LPS_deac-BSA neoglycoconjugate antigens

Neoglycoconjugates in carbonate buffer (50 mM, pH 9.2) were coated onto Maxisorb microtiter plates (96-well, U-bottom; Nunc, Wiesbaden, Germany) at 4℃ overnight. Antigen solutions were adjusted to equimolar concentrations of 50 pmol per well based on the amount of ligand present in the respective glycoconjugate. If not stated otherwise, 50 µl volumes were used. Plates were washed twice in PBS supplemented with Tween 20 (0.05%; Bio-Rad, Munich, Germany) and thimerosal (0.01%, PBS-T) and were then blocked with PBS-T supplemented with casein (2.5%, PBS-TC) for 1 h at 37℃ on a rocker platform followed by two washings. Appropriate serum dilutions in PBS-TC supplemented with 5% BSA (PBS–TCB) were added and incubated for 1 h at 37℃. After two washings, peroxidase-conjugated goat anti-bovine IgG [heavy- and light-chain specific (Dianova) diluted 1:1000 in PBS–TCB] was added and incubation was continued for 1 h at 37℃. The plates were washed three times with PBS-T. Commercial 3,3′,5,5′-tetramethylbenzidine (TMB; Uptima Interchim, Montluçon, France) substrate solution (100 µl) was added. After 30 min at 37℃, the reaction was stopped by the addition of aqueous oxalic acid (2%) and the plates were read by a microplate reader (Tecan Sunrise, Crailsheim, Germany) at 405 nm. Tests were run twice in quadruplicates with confidence values not exceeding 10%.

ELISA using LPS antigens

When LPS was used as an antigen in ELISA, microtiter polyvinyl plates (96-well, Falcon 3911; Becton Dickinson, Heidelberg, Germany) were coated with LPS (250 ng/well) diluted in PBS (pH 7.2) and were incubated overnight at 4℃. PBS and PBS-containing solutions were supplemented with thimerosal (0.01%). Further incubation steps were performed at 37℃ under gentle agitation. The coated plates were washed three times with PBS and blocked for 1 h at 37℃ with PBS supplemented with casein [2.5% (Sigma, Munich, Germany) PBS-C, 200 µl per well]. Serial serum dilutions in PBS-C were added subsequently and the mixture was incubated for 1 h at 37℃. After three washings in PBS, secondary Abs diluted in PBS-C (same source and dilution as above) were added and incubation was allowed for 1 h at 37℃. After three washings in PBS, substrate solution [freshly prepared and composed of azino-di-3-ethylbenzthiazolinsulfonic acid (1 mg) dissolved in substrate buffer (0.1 M sodium citrate, pH 4.5; 1 ml)] was added followed by the addition of hydrogen peroxide (25 µl of a 0.1% solution). After 30 min at 37℃, the reaction was stopped by the addition of aqueous oxalic acid (2%) and the plates were read by a microplate reader (Tecan Sunrise, Crailsheim, Germany) at 405 nm. Tests were run twice in quadruplicates with confidence values not exceeding 10%.

SDS-PAGE and Western blots

Proteinase K-digested whole cell lysates of E. coli O157 were applied to one large slot (12.5 cm) and separated by SDS-PAGE on a 5% stacking and 15% separating gel at a constant voltage of 150 V. The gels were transferred overnight onto polyvinylidene difluoride membranes (pore size 0.45 µm; Millipore, Schwalbach, Germany) by tank blotting (Bio-Rad, Munich, Germany). Prior to use, the membranes were wetted in methanol for 10 s, after which they were washed in distilled water for at least 5 min. Following transfer, the blots were cut into stripes (0.5 cm width) and placed in Mini-incubation trays (Bio-Rad, Munich, Germany). The following steps were performed at room temperature: after blocking in blotting buffer (50 mm Tris-HCl, 0.2 m NaCl, pH 7.4) supplemented with 10% non-fat dry milk for 1 h, the antisera diluted in blocking buffer were added, incubated overnight and washed 6 times (5 min each) in blotting buffer. Alkaline phosphatase-conjugated goat-anti bovine IgG (Fc-fragment specific; Dianova) was added (diluted 1:500 in blotting buffer) and incubation was continued for another 2 h. After washing as before, 5-bromo-4-chloro-3-indoylphosphate and p-toluidine p-nitroblue tetrazolium chloride (Bio-Rad, Munich, Germany) were added as substrates according to the manufactuer’s instructions. After 15 min the reaction was stopped by the addition of distilled water.

Results and discussion

Immunization of cows with LPS_deac-HC

In the first immunization, a total of 18 cows was immunized with LPS_deac-HC glycoconjugates containing the deacylated carbohydrate backbone of E. coli rough mutants of the R1, R2, R3, R4 or K-12 core type (complete core) or of the E. coli core-defective Rc-chemotype mutant J-5 (3 cows each), as described in the Materials and methods. Sera were collected over a period of 168 d and were then tested in LPS-ELISA and in conjugate-ELISA. As seen in Table 1, all but one animal (cow number 11) showed homologous seroconversion as determined by LPS-ELISA, which occurred 4 times on d 28, 11 times on d 56 and twice on d 140. All but one animal (cow number 33) showed homologous seroconversion as determined by conjugate-ELISA, which occurred 4 times on d 28, once on d 42 and 10 times on d 56. The serum of cow number 4 exhibited a high background activity with the same titers against the control antigen (glutardialdehyde activated BSA) and could thus not be included in the interpretation.

Table 1.

Observed seroconversion in cows after immunization with LPS_deac-HC glycoconjugate of E. coli rough mutants.

Immunogen	Cow no	Day after immunization where homologous and heterologous seroconversion was observed by ELISA using
		LPS of						LPS_deac-BSA conjugates of
		R1	R2	R3	R4	K-12	J-5	R1	R2	R3	R4	K-12	J-5
Ec R1	3	28 a	168	168	28	168		28	140	140	28	140
Ec R1	25	28						56			56	84
Ec R1	5	56						56	140	140	56	140	140
Ec R2	4		56		56	56	56	bga^b
Ec R2	24		56					140	56	140	140	56	140
Ec R2	6	28	56	56			140		56	56		56	56
Ec R3	33			140
Ec R3	23			56				56	56	56	56	56	56
Ec R3	7			56			56	140	140	42	140	140	140
Ec R4	2			140	56			84			28	140	140
Ec R4	22				28			56	140	140	28	140	56
Ec R4	8				56			56	140	140	56	140	140
Ec K-12	32					140						56
Ec K-12	20	140	140		126	56	140	140	56	140	28	56	140
Ec K-12	34		56	140	42	56	28	56	56	28		28	28
Ec J-5	31						56			56			56
Ec J-5	18						28	140	140	56	140	140	56
Ec J-5	11

Homologous reactivities printed in italics.

Data not valid owing to background activity with BSA alone.

This shows that the LPS_deac-HC conjugate is a potent immunogen resulting in the formation of Abs which bind equally to LPS and deacylated LPS and which are thus directed against carbohydrate epitopes of LPS.

Heterologous seroconversion was also observed in a total of 77 cases; 20 thereof were determined by LPS-ELISA and 57 by conjugate-ELISA. In most cases the heterologous seroconversion was observed later than the homologous titer rise. Table 2 shows the development of homologous titers of all animals over the observed period of 168 d. Only 4 of the 17 animals that had shown homologous seroconversion had titers below 10,000 as determined by LPS-ELISA; among these 4 were all 3 cows that had received the conjugate containing the LPS_deac of E. coli J-5 and 1 cow (number 34) that had been immunized with the E. coli K-12 glycoconjugate. Four animals had titers >100,000; among these were all 3 cows that had received the conjugate containing the LPS_deac of E. coli R4 and 1 cow (number 3) that had been immunized with the E. coli R1 glycoconjugate. The highest titer observed in conjugate-ELISA was 20,480 (animal number 3); 9 animals had titers between 1280 and 5120. From the 3 animals that had been immunized with the E. coli J-5 glycoconjugate none had titers >320.

Table 2.

Homologous Ab titers in cow sera after immunization with LPS_deac-HC glycoconjugate of E. coli rough mutants.

Cow no.	Immunogen	Titer by LPS-ELISA on d								Titer by conjugate ELISA on d
Cow no.	Immunogen	0 ^a	28	42	56	84	126	140	168	0	28	42	56	84	126	140	168
3	Ec R1	400	1600	1600	12,800	12,800	12,800	204,800	102,400	<20	160	160	1280	1280	1280	20,480	2560
25	Ec R1	200	800	800	12,800	12,800	6400	25,600	25,600	<20	20	20	2560	640	320	2560	1280
5	Ec R1	800	1600	800	12,800	3200	1600	6400	6400	<20	40	40	640	320	160	1280	640
4	Ec R2	100	400	800	6400	6400	1600	12,800	12,800	<20	<20	<20	bga^b	bga	bga	bga	bga
24	Ec R2	400	800	800	12,800	6400	3200	12,800	12,800	<20	<20	<20	320	320	160	640	640
6	Ec R2	1600	1600	1600	12,800	6400	3200	51,200	25,600	<20	<20	<20	640	160	80	2560	1280
33	Ec R3	1600	800	800	800	400	200	800	800	<20	<20	<20	20	<20	<20	20	<20
23	Ec R3	400	800	400	12,800	6400	800	6400	3200	<20	20	20	1280	1280	80	640	320
7	Ec R3	3200	1600	1600	6400	3200	800	12,800	3200	<20	40	40	640	640	1280	640	160
2	Ec R4	3200	6400	6400	12,800	6400	6400	409,600	204,800	<20	320	320	1280	640	320	5120	5120
22	Ec R4	800	3200	3200	51,200	25,600	12,800	204,800	102,400	<20	80	80	5120	2560	1280	2560	320
8	Ec R4	6400	6400	1600	51,200	12,800	6400	102,400	102,400	<20	<20	80	5120	1280	320	2560	1280
32	Ec K-12	3200	1600	1600	3200	1600	1600	12,800	12,800	<20	<20	20	320	320	160	640	320
20	Ec K-12	400	200	800	1600	3200	1600	12,800	6400	<20	20	20	640	160	160	1280	320
34	Ec K-12	1600	3200	1600	6400	3200	1600	6400	3200	<20	160	160	320	80	40	160	40
31	Ec J-5	200	200	200	1600	800	200	1600	800	<20	<20	<20	160	40	<20	160	20
18	Ec J-5	200	800	800	3200	1600	800	3200	1600	20	40	40	320	160	40	320	40
11	Ec J-5	400	800	400	800	800	400	800	400	<20	<20	<20	40	20	20	20	<20

Days of immunization are printed in italics.

Data not valid owing to background activity (BGA) with BSA alone.

At first glance absolute titers in LPS-ELISA seem to be higher than in conjugate-ELISA. However, in the former assay significant titers of up to 6400 were observed already in pre-immune sera whereas no reactivity except animal number 18 was observed in conjugate-ELISA.

The development of heterologous titers over the observed period of time is depicted in Table 3. Only one animal per immunogen is shown and only titers not differing by more than three titer steps from the highest homologous titer were considered significant. After immunization with the R1 conjugate (cow number 3) heterologous Abs against the R4 LPS were observed by conjugate- and LPS-ELISA. After immunization with the R2 conjugate (cow number 6) no heterologous Abs were observed by conjugate-ELISA. In LPS-ELISA significant titers against the R3 LPS were observed with only one titer step difference to the homologous titer. After immunization with the R3 conjugate (cow number 23), heterologous Abs against the R1, R4 and K-12 LPS were observed by conjugate-ELISA, and with R4 by LPS-ELISA. After immunization with the R4 or K-12 conjugate (cow numbers 2 and 20 respectively) no heterologous reactions were seen by conjugate- or LPS-ELISA. After immunization with the J-5 conjugate (cow number 18) heterologous reactions were detected with all LPS by conjugate-ELISA and with R3 and K-12 LPS by LPS-ELISA.

Table 3.

Heterologous Ab titers of cow sera after immunization with E. coli core oligosaccharides conjugated to hemocyanine (HC) as tested by conjugate ELISA (left) and LPS ELISA (right).

Cow no.	d^a	Immunogen	Final dilution yielding OD₄₀₅ >0.2 with indicated E. coli
			LPS_deac-BSA conjugate						LPS
			R1	R2	R3	R4	K-12	J-5	R1	R2	R3	R4	K-12	J-5
3	0	Ec R1	<20 ^b	<20	<20	<20	<20	<20	400	400	400	400	400	400
3	28		160	<20	<20	320	<20	<20	1600	400	200	1600	400	800
3	42	Ec R1	160	<20	<20	80	<20	<20	1600	400	200	1600	400	400
3	56		1280	<20	<20	160	<20	<20	12,800	400	200	3200	400	400
3	84		1280	<20	<20	320	<20	<20	12,800	800	400	6400	800	400
3	126	Ec R1	1280	<20	<20	320	<20	<20	12,800	800	400	3200	400	400
3	140		20,480	80	80	2560	80	40	204,800	800	400	51,200	400	400
3	168		2560	<20	20	640	20	20	102,400	1,600	800	12,800	1600	800
6	0	Ec R2	<20	<20	<20	<20	<20	<20	200	1600	1600	800	800	400
6	28		<20	<20	<20	<20	<20	<20	800	1600	3200	800	800	400
6	42	Ec R2	<20	<20	<20	<20	<20	<20	1600	1600	1600	800	1600	400
6	56		20	640	80	20	80	80	400	12,800	12,800	800	800	400
6	84		<20	160	40	<20	40	40	400	6400	6400	400	400	400
6	126	Ec R2	<20	80	<20	<20	<20	20	800	3200	6400	400	800	400
6	140		40	2560	80	20	80	160	400	51,200	25,600	400	800	1600
6	168		20	1280	80	20	40	80	400	25,600	12,800	200	800	800
23	0	Ec R3	<20	<20	<20	<20	<20	<20	200	200	400	400	200	400
23	28		<20	<20	20	<20	20	20	400	400	800	400	400	400
23	42	Ec R3	<20	<20	20	<20	20	20	200	200	400	800	400	400
23	56		320	80	1280	160	320	160	400	200	12,800	800	400	6400
23	84		160	40	1280	40	320	320	400	200	6400	400	800	800
23	126	Ec R3	20	20	80	40	20	40	200	400	800	400	400	800
23	140		80	40	640	80	80	160	200	400	6400	400	800	800
23	168		40	20	320	20	40	40	200	400	3200	400	800	400
2	0	Ec R4	<20	<20	<20	<20	<20	<20	1600	800	800	3200	800	400
2	28		40	<20	<20	320	<20	<20	1600	800	800	6400	800	400
2	42	Ec R4	40	<20	<20	320	<20	<20	1600	400	800	6400	800	800
2	56		40	<20	<20	1280	<20	<20	1600	400	800	12,800	800	800
2	84		80	<20	<20	640	<20	<20	1600	800	800	6400	800	800
2	126	Ec R4	20	<20	<20	320	<20	<20	1600	800	400	6400	800	800
2	140		160	40	40	5120	80	80	1600	800	1600	409,600	800	800
2	168		40	20	20	5120	40	20	800	800	800	204,800	400	400
20	0	Ec K-12	<20	<20	<20	40	<20	<20	200	800	200	400	400	200
20	28		<20	<20	<20	160	20	20	200	800	200	400	200	200
20	42	Ec K-12	<20	<20	<20	80	20	<20	200	400	100	400	800	200
20	56		<20	160	<20	160	640	40	400	800	100	400	1600	200
20	84		<20	40	<20	160	160	40	200	400	100	400	3200	200
20	126	Ec K-12	<20	20	<20	80	160	40	100	800	200	6400	1600	100
20	140		160	160	80	640	1280	320	800	1600	200	400	12,800	400
20	168		40	40	<20	160	320	80	400	800	200	200	6400	200
18	0	Ec J-5	<20	<20	<20	<20	<20	20	400	<100	800	200	800	200
18	28		<20	<20	<20	<20	<20	40	800	<100	800	200	1600	800
18	42	Ec J-5	<20	<20	<20	<20	<20	40	400	100	1600	100	800	800
18	56		<20	<20	80	<20	20	320	200	100	800	100	800	3200
18	84		<20	<20	40	<20	<20	160	400	100	800	100	800	1600
18	126	Ec J-5	40	40	???	20	20	40	200	100	800	100	1600	800
18	140		320	80	320	160	320	320	200	<100	1600	100	1600	3200
18	168		40	20	40	20	40	40	200	100	800	100	800	1600

Days of immunization are printed in italics.

Homologous titers are printed in bold.

Core-typing of clinical E. coli mastitis strains

As it was planned for the next step to design a vaccination protocol in which cows should be immunized with an LPS_deac-HC glycoconjugate vaccine and be challenged with an E. coli wild type strain, we first determined which core type occurs most frequently in clinical isolates from bovine mastitis. Fifty isolates were typed using monoclonal Abs against the different E. coli core types, as described in the Materials and Methods. Twenty strains (40%) had the R1 core, 17 strains (34%) the R2 core, 11 strains (22%) the R3 core and two strains (4%) the R4 core. The K-12 core was not detected in any of the clinical isolates. These results are in accordance with those published.²⁶

Immune response to a mixture of glycoconjugate vaccines

We also investigated whether a mixture of conjugates containing the LPS_deac of all five E. coli core types was effective. As seen in Table 4, three out of four animals responded to the mixture of immunogens with seroconversion against all five antigens independent of whether the sera were tested in LPS-ELISA or in conjugate-ELISA. The highest titers in LPS- and conjugate-ELISA ranged from 800 to 102,400 and from 80 to 40,960 respectively.

Table 4.

Ab titers of cow sera after immunization with LPS_deac-HC glycoconjugate containing a mixture all E. coli core types.

Cow no.	d	Final dilution yielding OD₄₀₅ >0.2 with E. coli
		LPS					LPS_deac-BSA
		R1	R2	R3	R4	K-12	R1	R2	R3	R4	K-12
3161	0	800	<100	100	400	100	80	<20	<20	20	<20
	28	51,200	400	6400	12,800	1600	2560	40	320	1280	320
	42	102,400	400	6400	25,600	1600	5120	80	640	2560	640
	56	51,200	800	6400	25,600	51,200	40,960	1280	2560	5120	2560
3167	0	200	<100	200	100	100	20	<20	<20	<20	<20
	28	1600	200	200	800	1600	320	80	40	160	160
	42	3200	100	200	1600	800	320	40	20	160	160
	56	6400	800	400	3200	6400	1280	320	160	640	640
3148	0	200	<100	100	200	200	20	<20	<20	<20	<20
	28	800	100	200	400	400	40	<20	<20	40	20
	42	800	200	200	400	400	<20	<20	<20	20	20
	56	400	200	800	800	400	40	<20	20	80	40
3154	0	400	100	400	400	800	20	<20	<20	40	20
	28	6400	200	1600	6400	3200	640	40	40	320	320
	42	6400	200	800	6400	1600	320	40	20	320	160
	56	12,800	1600	800	6400	6400	2560	320	80	640	640

Clinical vaccination challenge study

In the last experiment, we immunized cows in groups of 12 animals with LPS_deac-HC glycoconjugate of E. coli R1, or a mixture of R1, R2 and R3 on d 0, 42 and 84; a control group of 12 animals without immunization was included. Two wk later, the animals were challenged with an E. coli wild type strain with the R1 core type. Sera were collected over a period of 119 d and tested by LPS- or conjugate-ELISA using LPS or LPS_deac of E. coli R1, R2 or R3 as antigen. Table 5 summarizes the results with regard to seroconversion whereby those titer rises occurring during the vaccination phase and those which were the result of the renewed contact with antigen by the challenge are differentiated. No seroconversion was observed after vaccination in the control group. After the challenge, seroconversion was observed against the R1 core type in 7 and 10 cases by LPS- and conjugate-ELISA respectively. Two cows showed seroconversion against the R2 core type and one animal against the R3 core type; however, these were only measured by conjugate-ELISA and not by LPS-ELISA.

Table 5.

Seroconversion of cows immunized with LPS_deac-HC glycoconjugate of E. coli R1 or a mixture of R1, R2 and R3, and challenged with an E. coli wild type strain with the R1 core type.

Group	Number of animals with seroconversion after
	vaccination determined by ELISA with:						challenge determined by ELISA with:
	E. coli LPS			E. coli LPS_deac-BSA			E. coli LPS			E. coli LPS_deac-BSA
	R1	R2	R3	R1	R2	R3	R1	R2	R3	R1	R2	R3
Control							7			10	2	1
R1, R2, R3	8	4	2	12	12	10	6	2	1	5	0	1
R1	8	3	0	11	9	9	4	1	0	6	0	0

In the group immunized with the mixture of R1, R2 and R3 conjugates, 12, 12 and 10 animals showed seroconversion in conjugate-ELISA against R1, R2 and R3, respectively, and 8, 4 and 2 animals, respectively, when Abs were measured by LPS-ELISA. After challenge, a second seroconversion was observed, most often against the R1 core type (for details see Table 5). The results obtained in the R1 group were similar to those obtained for the mixture group.

Clinical evaluation showed that all cows developed clinical mastitis within 12 h of challenge, as evidenced by increased rectal temperature, increased somatic cell counts and the presence of E. coli in milk. The morning after challenge rectal temperatures peaked in all 3 groups; the average increase was 2.2 ± 0.5, 2.5 ± 0.6 and 2.6 ± 0.5℃ for the groups vaccinated with the mixture of (R1, R2 and R3), with R1 alone or with the adjuvant only respectively. Thereafter, the rectal temperatures of all groups decreased.

On the first day after challenge E. coli was isolated from the milk of all challenged quarters from the three groups. Thereafter, the number of quarters positive for E. coli decreased gradually; on day 7 E. coli was isolated from 4 out of 12, 6 out of 11 and 4 out of 12 quarters of groups immunized with the mixture (R1, R2 and R3), with R1 alone or with the adjuvant respectively. On d 14 this was 2 out of 12, 3 out of 11 and 2 out of 12 quarters for the 2 vaccinated groups and the placebo group respectively. The average colony counts of E. coli per group is shown in Figure 1A. These data show that there is no beneficial effect of vaccination on the average colony counts of E. coli in milk.

Figure 1.

Average bacterial counts (A) (cfu/ml) and average somatic cell counts (SCC) (B) of 12 cows each vaccinated with E. coli R1 conjugate (triangles), E. coli R1, R2, R3 mixture conjugate (squares) or receiving a placebo (diamonds) and challenged 2 wk after the last immunization with E. coli O157 (R1 core type). Samples were taken during the morning (am) and in the afternoon (pm).

The somatic cell counts (SCC) of all challenged quarters increased after challenge. At 24 h after challenge the average SCC was 10^6.9cells/ml for all 3 groups. Average somatic cell counts remained high up to d 21 (day of last observations, see Figure 1B). No beneficial effect of vaccination on the level of SCC was observed.

To understand the failure of the vaccination we analyzed sera from nine cows vaccinated with the R1 vaccine by Western blots to see whether Abs against the rough and smooth fraction of the E. coli O157 LPS were present. As seen in Figure 2, Abs against the rough fraction were detected in all cows at dilutions of up to 1:10,000 whereas no Abs against the smooth fraction were found even at the lowest dilution of 1:100. This shows that the vaccination did not lead to the formation of Abs similar to the broadly cross-reactive Ab WN1 222-5.²³

Figure 2.

Western blots of proteinase K-digested whole cell lysates of E. coli O157 separated by SDS-PAGE on a 15% separating gel. Blots were developed with sera collected 14 wk after the first immunization at different dilutions (a, 1:100; b, 1:1,000; c, 1:10,000).

The analysis of the clinical data led to the conclusion that vaccination did not prevent, or reduce, the intramammary infection after challenge with E. coli.

Conclusions

The present study gives insight into the bovine immune response to carbohydrate epitopes in LPS of E. coli rough mutants. Cows, as many other mammalians, produce anti-LPS Abs after immunization with whole bacteria, but it has been shown earlier that the immunization with a deep rough (Re-type) bacterium of E. coli K-12 failed to protect cows from intramammary challenge with an E. coli wild type strain.¹⁵

This study focused on the immune response against complete core oligosaccharides from the five different core types of E. coli as it has been shown that the mAb WN1 222-5 could be induced in mice which bound equally to all core types of E. coli and, at the same time, bound also to all E. coli wild type strains.²³ After immunization with glycoconjugates representing all core types the immune response was directed mainly against the homologous immunizing antigen with a low degree of cross-reactivity. Therefore, Abs with similar binding properties as WN1 222-5 could not be induced.

Nevertheless, we investigated whether the homologous Abs would protect cows against the intramammary challenge with an E. coli wild type strain with the same core type. The vaccination experiment (carried out on a total of 36 cows) showed that neither the vaccination with R1 core type alone nor with a mixture of all clinically relevant core types (R1, R2 and R3 core) failed to protect from intramammary challenge with E. coli wild type strain O157 with the R1 core, although the animals developed high titers against the R1 core.

We conclude that deacylated LPS conjugated to hemocyanine cannot be used as a vaccine to prevent bovine mastitis.

Footnotes

Acknowledgment

We thank U. Agge, S. Cohrs, I. von Cube, C. Schneider and V. Susott for technical assistance.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors do not have any potential conflicts of interest to declare.

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Evaluation of a LPS-based glycoconjugate vaccine against bovine Escherichia coli mastitis: Formation of LPS Abs in cows after immunization with E. coli core oligosaccharides conjugated to hemocyanine

Abstract

Keywords

Introduction

Materials and methods

Bacterial LPS and neoglycoconjugates

Immunization with LPSdeac-HC glycoconjugates

Vaccination and challenge

Clinical measurements

Core typing of clinical bovine mastitis isolates

Serology

ELISA using LPSdeac-BSA neoglycoconjugate antigens

ELISA using LPS antigens

SDS-PAGE and Western blots

Results and discussion

Immunization of cows with LPSdeac-HC

Core-typing of clinical E. coli mastitis strains

Immune response to a mixture of glycoconjugate vaccines

Clinical vaccination challenge study

Conclusions

Footnotes

Acknowledgment

Funding

Conflict of interest

References

Immunization with LPS_deac-HC glycoconjugates

ELISA using LPS_deac-BSA neoglycoconjugate antigens

Immunization of cows with LPS_deac-HC