Advertisement

Case Study: Combined risk factors and digestive disorders in mid-lactation Holstein cows

      ABSTRACT

      Objective

      Digestive disorders can be a significant cause of disease in dairies and are frustrating because of their unpredictability. There is limited research on these conditions, and experimental exposure to individual risk factors often fails to cause disease. In this case study, we document the outbreak and resolution of digestive disorders among 15 control cows enrolled in a larger study.

      Materials and Methods

      Over 14 wk, cows were individually fed, with milk yield and composition, blood variables, and health observations recorded. The diet included drought-stressed corn silage that introduced difficulties including low energy density, high DM content (making it unstable at feed-out), and mycotoxin contamination.

      Results and Discussion

      By wk 4 to 5 of the study, fecal consistency decreased and milk fat content had dropped from 3.7% (±0.20) to 3.4% (±0.20), on average. Coincident with the onset of environmental heat stress, 3 cows developed severe digestive disorders, resulting in a displaced abomasum in one cow. At that point, the diet was changed to replace some corn silage with wheat straw, a direct-fed microbial was added to the diet, and organic acid treatment of the silage face was initiated. Within a month after these changes were implemented, essentially all signs of digestive problems resolved, including milk fat content, fecal consistency, and blood plasma concentrations of haptoglobin and d-lactate, although milk yield decreased.

      Implications and Applications

      This case study points to multiple factors that likely combined to lead to microbial and gastrointestinal disruptions resulting in clinical disease in a subset of cows.

      Key words:

      INTRODUCTION

      Digestive disorders occur among lactating cows in dairy farms. These sporadically occurring challenges, ranging from mild diarrhea to hemorrhagic bowel syndrome (
      • Owaki S.
      • Kawabuchi S.
      • Ikemitsu K.
      • Shono H.
      • Furuoka H.
      Pathological findings of hemorrhagic bowel syndrome (HBS) in six dairy cattle cases..
      ), rarely offer an obvious cause, leaving dairy producers confounded. Digestive challenges are believed to be multifaceted in their development (
      • Kirkpatrick M.
      • Timms L.
      • Kersting K.W.
      • Kinyon J.
      Case study—Jejunal hemorrhage syndrome in dairy cattle..
      ). Opportunistic gastrointestinal pathogens (
      • Godden S.
      • Frank R.
      • Ames T.
      Survey of Minnesota dairy veterinarians on the occurrence of and potential risk factors for jejunal hemorrhage syndrome in adult dairy cows..
      ;
      • Dennison A.C.
      • VanMetre D.C.
      • Callan R.J.
      • Dinsmore P.
      • Mason G.L.
      • Ellis R.P.
      Hemorrhagic bowel syndrome in dairy cattle: 22 cases (1997–2000)..
      ), excessive flow of fermentable carbohydrate or protein to the hindgut (
      • Gressley T.F.
      • Hall M.B.
      • Armentano L.E.
      Ruminant Nutrition Symposium: Productivity, digestion, and health responses to hindgut acidosis in ruminants..
      ), and the presence of mycotoxins (
      • Grenier B.
      • Applegate T.J.
      Modulation of intestinal functions following mycotoxin ingestion: Meta-analysis of published experiments in animals..
      ) in the diet are just some of the known causative agents of digestive disorders. These conditions are thought to contribute to a disruption of the gut barrier (i.e., “leaky gut”;
      • Kvidera S.K.
      • Dickson M.J.
      • Abuajamieh M.
      • Snider D.B.
      • Fernandez M.V.S.
      • Johnson J.S.
      • Keating A.F.
      • Gorden P.J.
      • Green H.B.
      • Schoenberg K.M.
      • Baumgard L.H.
      Intentionally induced intestinal barrier dysfunction causes inflammation, affects metabolism, and reduces productivity in lactating Holstein cows..
      ), which in turn leads to bacterial invasion and dramatic inflammation of the tissue (
      • Godden S.
      • Frank R.
      • Ames T.
      Survey of Minnesota dairy veterinarians on the occurrence of and potential risk factors for jejunal hemorrhage syndrome in adult dairy cows..
      ;
      • Kirkpatrick M.
      • Timms L.
      • Kersting K.W.
      • Kinyon J.
      Case study—Jejunal hemorrhage syndrome in dairy cattle..
      ;
      • Dennison A.C.
      • VanMetre D.C.
      • Callan R.J.
      • Dinsmore P.
      • Mason G.L.
      • Ellis R.P.
      Hemorrhagic bowel syndrome in dairy cattle: 22 cases (1997–2000)..
      ). Previous research into this topic has been limited, as studies have assessed individual risk factors, which have been inadequate to replicate the digestive disorders on their own (
      • Ewoldt J.M.
      • Anderson D.E.
      Determination of the effect of single abomasal or jejunal inoculation of Clostridium perfringens type A in dairy cows..
      ;
      • Gressley T.F.
      • Davison K.A.
      • Macies J.
      • Leonardi C.
      • McCarthy M.M.
      • Nemec L.M.
      • Rice C.A.
      Effect of abomasal carbohydrates and live yeast on measures of postruminal fermentation..
      ).
      Gastrointestinal disease on commercial dairy operations may typically result from multiple risk factors occurring simultaneously. Documentation of these factors, throughout the course of an outbreak, would help to improve our understanding of the disease process, but commercial farms rarely obtain individual cow data or longitudinal samples that would allow for analysis of contributing factors before disease onset.
      In the process of conducting a controlled nutrition study, the authors observed the onset and resolution of gastrointestinal disease. For experimental purposes, samples of feed, milk, blood plasma, and feces were collected that have allowed us to document relevant changes that likely contributed to disease progression and resolution over the course of 14 wk. Our objective in this case study was to describe our observations and evaluate plausible explanations for the disease process.

      MATERIALS AND METHODS

      All protocols were approved by the Kansas State University Animal Care and Use Committee. Fifteen multiparous (10 in lactation 2 and 5 in lactation 3+) Holstein dairy cows (94–197 DIM) were control animals included in a larger production study carried out between April 30 and August 5, 2019. Cows were housed at the Kansas State University Dairy Teaching and Research Center (Manhattan, KS) in tie-stalls with rubber mats and wood shavings. A TMR (Table 1) was prepared once daily, with feed delivered to cows twice daily (0630 and 1830 h); the TMR consisted of corn silage, alfalfa hay, corn byproduct (Sweet Bran, Cargill), whole cottonseed, and a grain mix. Kansas experienced drought conditions during the summer of 2018, which resulted in the corn silage having low starch content and greater-than-normal NDF digestibility (Table 2). The low energy content of silage required greater concentrate levels to meet predicted energy requirements for highly productive mid-lactation cows. Feed and feed-refusal samples were collected 3 times per week, composites representing 2-wk periods, and sent to Cumberland Valley Analytical Services for chemical composition analysis. Dry matter intake was determined by recording daily feed intake and factoring in TMR DM content, updated weekly.
      Table 1Ingredient and chemical composition of TMR (% of DM except where noted)
      ItemWk 1–8Wk 9–14
      Ingredients
       Corn silage A16.76.29
       Corn silage B6.29
       Alfalfa hay20.820.9
       Corn milling product
      Sweet Bran, Cargill.
      17.417.5
       Cottonseed2.792.81
       Corn grain24.825.0
       Expeller soybean meal
      Soy Plus, Landus Cooperative.
      12.212.2
       Limestone1.251.26
       Sodium bicarbonate0.850.86
       Calcium salts of long-chain

        fatty acids
      Essentiom, Arm & Hammer Animal Nutrition.
      0.830.84
       Micronutrient premix2.542.44
       Wheat straw3.51
       Direct-fed microbial
      Biofix Plus Pro, Biomin America.
      0.11
      Nutrients
       DM, % as fed61.759.9
       NEl, Mcal/kg of DM1.691.63
       CP18.519.4
       Ether extract5.035.01
       NDF (aNDF
      aNDF = NDF after amylase treatment.
      )
      31.431.3
       ADF20.320.9
       Ash8.469.72
       Ca1.001.25
       P0.490.50
      1 Sweet Bran, Cargill.
      2 Soy Plus, Landus Cooperative.
      3 Essentiom, Arm & Hammer Animal Nutrition.
      4 Biofix Plus Pro, Biomin America.
      5 aNDF = NDF after amylase treatment.
      Table 2Chemical composition of corn silage sources (% DM basis except where noted)
      ItemCorn

      silage A
      Corn

      silage B
      DM, % as fed42.529.1
      CP9.210.7
      ADF25.626.0
      aNDF
      aNDF = NDF after amylase treatment.
      41.445.9
      NDF digestibility, 30 h,

       % of aNDF
      56.661.6
      Lignin2.72.6
      Starch28.118.4
      Crude fat3.23.6
      Ash5.56.3
      Calcium0.30.3
      Phosphorus0.20.3
      Magnesium0.20.2
      Potassium0.91.6
      1 aNDF = NDF after amylase treatment.
      Cows were milked 3 times daily at 0400, 1100, and 1800 h. Milk yield was recorded electronically at each milking. Milk samples were collected for 6 consecutive milkings each week and sent to the Miner Institute for analysis of fat, true protein, lactose (B-2000 Infrared Analyzer; Bentley Instruments), and fatty acid class by Fourier-transform mid-infrared spectroscopy (Delta Combi 300). After analysis, data were composited by week. Energy-corrected milk yield was calculated as (0.327 × milk yield) + (12.95 × fat yield) + (7.65 × protein yield) and feed efficiency was calculated as ECM/DMI.
      Blood samples were collected from the coccygeal vein once every other week at 1500 h into 2 evacuated 10-mL tubes containing potassium EDTA (Vacutainer; Becton, Dickinson and Co.). Blood was put on ice immediately after collection and then centrifuged at 2,415 × g (Beckman J06B Centrifuge) for 15 min. Plasma was collected and stored at −20°C for later analysis. Plasma was analyzed for haptoglobin (
      • Cooke R.F.
      • Arthington J.D.
      Concentrations of haptoglobin in bovine plasma determined by ELISA or a colorimetric method based on peroxidase activity..
      ), d-lactate (Colorimetric Assay Kit; Biovision Inc.), serum amyloid A (SAA; ELISA Assay kit; Tridelta Development Ltd.), lipopolysaccharide binding protein (LBP; ELISA Assay Kit; Cell Sciences Inc.;
      • Bannerman D.D.
      • Paape M.J.
      • Hare W.R.
      • Sohn E.J.
      Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge..
      ), and diamine oxidase (DAO; Biovision Inc.).
      Over the course of the study, 5 of the 15 cows were removed from the study for health concerns. Some loose manure was noted relatively early in the study, particularly from wk 4 on; however, for most cows and most days, manure consistency was within the normal range.
      The first cow to be removed from study was in her fourth lactation and developed hock inflammation in wk 4. The next 3 cows were removed from the study for digestive disorders. During wk 5, the first serious summer heat stress window occurred, with mean weekly environmental temperature-humidity index (THI) climbing past 70 (Figure 1). At the end of wk 5, a second-lactation cow suddenly (within 24 h) went off feed and had a sharp decrease in milk production (from a mean of 38 kg/d down to 19.5 kg/d). Physical examination revealed extremely high rumen motility, but her body temperature and water intake were normal. Within 48 h, she was diagnosed with a displaced abomasum. In wk 7, 2 more second-lactation cows were removed from the study within 48 h of each other, after 4 d of declining feed intake. Digestive tract abnormalities (high motility, loose manure) were again observed, and one of these cows also showed some apparent neurological problems.
      Figure 1
      Figure 1Mean external maximum daily temperature (MaxTemp, °F), minimum daily temperature (MinTemp, °F), relative humidity (RH, %), and temperature-humidity index (THI) by week of the study. The double orange line indicates the THI level associated with negative effects on dairy cow physiology. Temperature in °C = 5/9 × (°F − 32).
      At this point in the study (wk 7), composite samples (representing 2-wk periods) collected in the initial 6 wk of the study were sent for initial mycotoxin analysis (Romer Labs Inc.; LC-MS), and the diet was adjusted to partially replace some excessively dry corn silage (corn silage A) with another corn silage (corn silage B) and some straw to enhance physically effective fiber content of the diet. Furthermore, we began treating the approximately 5.8 m2 of silage faces with 2 L/d of organic acids (0.34 L/m2 per d; Ultra-Curb, Kemin) to limit mold growth at feed-out and added a direct-fed microbial (Biofix Plus Pro; Biomin America) to the grain mix at 0.10% of the ration (DM basis) with the intention of limiting mycotoxin bioavailability. All of these changes were in place by the end of wk 8. One additional cow (third lactation) was removed from study during wk 13 due to clinical mastitis. Although we no longer sampled cows that were removed from the study, all cows recovered and remained in the herd. Twice during the study (wk 9 and 14), feed and fecal samples were collected to enumerate viable clostridia bacteria and those from the species C. perfringens specifically.
      To assess what response variables might have changed in a manner that coincided with the observed health problem, we assessed whether variables deviated from the baseline (wk 1) observations over time. Time series data were analyzed using SAS 9.4 (SAS Institute Inc.). Data were modeled with the Mixed Procedure using week, parity group (2 vs. 3+), and week × parity group interaction as fixed effects with cow as a random effect. Repeated measures over time were modeled with a heterogeneous autoregression covariance structure. Conditional Studentized residuals were used to check for normality and to remove outliers (>4 or <−4). Significant effects were declared at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. All data are expressed as LSM and SEM. Differences between wk 1 and subsequent weeks were evaluated by the pdiff option of the Mixed Procedure when the overall effect of week was significant.

      RESULTS AND DISCUSSION

      Dry matter intake and milk yield (Figure 2) were greater for cows in parity 3+ versus parity 2 (P = 0.01) and both varied by week (P < 0.01) across parities. Dry matter intake increased in wk 2 and 4 relative to wk 1 (P < 0.05), whereas milk yield was greater in wk 2, followed by lesser production in wk 10 to 14 relative to wk 1 (P < 0.05). Parity groups did not differ in milk fat content (P = 0.47, Figure 2C); however, there was week-to-week variation (P = 0.01). Fat content was decreased in wk 2 through 7, 9, 10, and 13 relative to wk 1 (P < 0.05) before recovering by wk 14. Second-lactation cows tended to have greater milk protein content compared with older cows (P = 0.098, Figure 2D); in addition, wk 2, 4, 5, 7, 8, and 10 through 14 all differed from wk 1 (P < 0.05). Energy-corrected milk yield was greater in parity 3+ cows versus second-lactation cows (P = 0.02; Figure 2E), and wk 2 as well as wk 9 through 14 differed from wk 1 (P < 0.05). Feed efficiency (defined as ECM yield/DMI) is shown in Figure 2F. Parity did not affect feed efficiency (P = 0.92), but efficiency was decreased in wk 4 through 14 versus wk 1 (P < 0.05). In addition to traditional milk components, we had data on classes of milk fatty acids, potentially providing insight into specific milk fat synthesis processes that may have been disrupted during the health challenge. Yields of short- and medium-chain fatty acids derived from mammary de novo synthesis (Figure 3A), mixed-source (Figure 3B), and long-chain fatty acids derived from circulating fatty acid pools (Figure 3C) all showed reductions over time. However, de novo synthesized fatty acid yields did not decline (relative to wk 1) until wk 9 onward, whereas preformed fatty acid yields had already declined by wk 4, suggesting that mammary uptake of fatty acids from blood—or possibly absorption of dietary fatty acids—was disrupted before declines in de novo synthesis.
      Figure 2
      Figure 2Productivity of cows through a spontaneous digestive disease outbreak. (A) Dry matter intake; (B) milk production; (C) milk fat concentration; (D) milk protein concentration; (E) ECM yield; and (F) feed efficiency among cows in parity 2 or 3+. Values are means ± SE. #P < 0.05 versus wk 1.
      Figure 3
      Figure 3Fatty acid yields of cows through a spontaneous digestive disease outbreak. (A) De novo synthesized (<C16) fatty acids; (B) mixed-source (C16) fatty acids; and (C) pre-formed (>C16) fatty acid yields of cows in parity 2 or 3+. Values are means ± SE. #P < 0.05 versus wk 1.
      As a case study, interpretation of these observations must be carried out cautiously. Multiple factors were changing simultaneously during the study, including weather conditions, microbial and mycotoxin contaminants, forage sources, and mitigation strategies. The uptick in feed consumption through wk 4 likely reflected adaptation to the new diet, whereas variations after that time likely reflect a combination of responses to heat stress and advancing stage of lactation. Although a progressive decrease in milk yield is expected in this group of cows past peak lactation (
      • DePeters E.J.
      • Smith N.E.
      • Acedo-Rico J.
      Three to two times daily milking of older cows, and first lactation cows for entire lactations..
      ), the more dramatic decline in milk yield from wk 9 through 14 was likely in part a response to the change in diet, particularly the inclusion of dietary straw to increase effective fiber content. Previous research has shown that adding slowly fermenting fiber to lactation diets typically decreases DMI and fluid milk yield (
      • Li C.
      • Beauchemin K.A.
      • Yang W.
      Feeding diets varying in forage proportion and particle length to lactating dairy cows: I. Effects on ruminal pH and fermentation, microbial protein synthesis, digestibility, and milk production..
      ).
      It is well established that there is a negative effect of heat stress on feed intake and production in ruminants (
      • Ominski K.H.
      • Kennedy A.D.
      • Wittenberg K.M.
      • Moshtaghi Nia S.A.
      Physiological and production responses to feeding schedule in lactating dairy cows exposed to short-term, moderate heat stress..
      ;
      • West J.W.
      Effects of heat-stress on production in dairy cattle..
      ;
      • Allen J.D.
      • Hall L.W.
      • Collier R.J.
      • Smith J.F.
      Effect of core body temperature, time of day, and climate conditions on behavioral patterns of lactating dairy cows experiencing mild to moderate heat stress..
      ;
      • Polsky L.
      • von Keyserlingk M.A.G.
      Invited review: Effects of heat stress on dairy cattle welfare..
      ). The THI threshold for negative effects on a dairy cow is around 68 (
      • St. Pierre N.R.
      • Cobanov B.
      • Schnitkey G.
      Economic losses from heat stress by US livestock industries..
      ;
      • Zimbleman R.B.
      • Rhoads R.P.
      • Baumgard L.H.
      • Collier R.J.
      Revised temperature humidity index (THI) for high producing dairy cows..
      ). Throughout the study, mean ambient THI levels (regional weather station) were consistently close to or above this threshold (Figure 1); although the barn housing the cows employed evaporative cooling to combat heat stress, it was not completely prevented during the heat of the day for most days after wk 4.
      • Kadzere C.T.
      • Murphy M.R.
      • Silanikove N.
      • Maltz E.
      Heat stress in lactating dairy cows: A review..
      demonstrated that there is a correlation between milk production and metabolic heat load, and recent research has postulated that the THI threshold for heat stress needs to be modified to match individual animal traits such as genetics and milk production (
      • Sanchez J.P.
      • Misztal I.
      • Aguilar I.
      • Zumbach B.
      • Rekaya R.
      Genetic determination of the onset of heat stress on daily milk production in the US Holstein cattle..
      ;
      • Hammami H.
      • Vandenplas J.
      • Vanrobays M.-L.
      • Rekik B.
      • Bastin C.
      • Gengler N.
      Genetic analysis of heat stress effects on yield traits, udder health, and fatty acids of Walloon Holstein cows..
      ).
      Mycotoxin concentrations detectable in the TMR and ingredients throughout the study are presented in Table 3 and Figure 4. Total trichothecene (nivalenol + deoxynivalenol) concentrations in the diet stayed between 1,327 and 1,735 μg/kg (DM basis) in TMR samples collected throughout the study. Zearalenone concentrations in TMR samples hovered around the detection limit of 81.4 μg/kg (DM basis), with a peak concentration of 171.4 μg/kg detected. There is relatively little evidence available to establish concentrations of mycotoxins that are of concern for ruminants. There is relatively more information regarding responses to aflatoxins, but we did not detect that class of compounds in any sample analyzed. Fusarium fungi produce trichothecenes including nivalenol and deoxynivalenol (DON, also known as vomitoxin), the 2 compounds detected in our samples. Zearalenone is also produced by Fusarium fungi but is an estrogenic metabolite. On their own, these mycotoxins present challenges to producers, but previous research has shown that exposure to mixtures of mycotoxins can have a more acute effect (
      • Alassane-Kpembi I.
      • Schatzmayr G.
      • Taranu I.
      • Marin D.
      • Puel O.
      • Oswald I.P.
      Mycotoxins co-contamination. Methodological aspects and biological relevance of combined toxicity studies..
      ; Feijó Correa et al., 2018;
      • Yang L.
      • Tu D.
      • Wu Y.
      • Liu Y.
      • Hu Y.
      • Liu T.
      • Tan L.
      • Li Y.
      • Lei H.
      • Zhan Y.
      • Wang N.
      • Deng Z.
      • Guo S.
      • Wang A.
      Distribution and persistence of residual T-2 and HT-2 toxins from moldy feed in broiler chickens..
      ).
      Table 3Mycotoxin concentrations (range, n = 3–4 per phase) of diet and ingredients (as-fed basis)
      ND = below detection limit.
      ItemNivalenol,
      Limit of detection = 100 μg/kg.
      μg/kg
      Deoxynivalenol,
      Limit of detection = 100 μg/kg.
      μg/kg
      Fumonisin B1,
      Limit of detection = 0.1 mg/kg.


      mg/kg
      Zearalenone,
      Limit of detection = 51.7 μg/kg.
      μg/kg
      Ochratoxin A,
      Limit of detection = 1.1 μg/kg.
      μg/kg
      Wk 1–8Wk 9–14Wk 1–8Wk 9–14Wk 1–8Wk 9–14Wk 1–8Wk 9–14Wk 1–8Wk 9–14
      TMR611–755170–295197–441630–729ND–0.30.6–1.1ND–7678–110NDND
      Ingredient
       Grain mixNDNDND–488ND–3471.2–7.51.6–2.7ND–253ND–78NDND
       Corn silage A1,088–1,652423–682NDND0.2–0.50.4–0.5NDNDND–4.2ND–3.0
       Corn silage B1,201–1,235NDNDNDND
       Sweet Bran
      Sweet Bran, Cargill.
      1,642–3,0121,833–3,3702,780–7,9661,201–2,4301.7–2.71.9–2.4619–665513–580NDND
       Alfalfa hayND–264160–289NDND–120NDNDNDNDNDND
       CottonseedNDNDND–133ND–155NDNDNDNDNDND
       Wheat strawND–175110–5,111NDND–60ND
      1 ND = below detection limit.
      2 Limit of detection = 100 μg/kg.
      3 Limit of detection = 100 μg/kg.
      4 Limit of detection = 0.1 mg/kg.
      5 Limit of detection = 51.7 μg/kg.
      6 Limit of detection = 1.1 μg/kg.
      7 Sweet Bran, Cargill.
      Figure 4
      Figure 4Mycotoxin concentration of composite ration samples (DM basis). Values are means, with trichothecenes being the summation of nivalenol and deoxynivalenol concentrations. **Weeks where zearalenone concentrations were below the detection limit of 84.1 μg/kg (DM basis).
      Many studies have now shown that exposing cultured intestinal epithelial cells to mycotoxins can cause cytotoxicity and also induce production of inflammatory mediators in these cells (
      • Pinton P.
      • Oswald I.P.
      Effect of deoxynivalenol and other type B trichothecenes on the intestine: A review..
      ). Although it is difficult to translate from dietary concentrations to corresponding concentrations expected in digesta through the ruminant gastrointestinal tract, we can come up with approximations. Assuming that trichothecenes are not absorbed or degraded in the gut, that diet DM digestibility is approximately 60%, and that hindgut digesta is about 10% DM, TMR concentrations of 500 μg/kg (as-fed basis) would correspond to a DON concentration in the distal gut of approximately 1 µM. On their own, nivalenol and DON had subtle inflammatory effects on porcine jejunal explants at these concentrations; however, the synergistic effects of co-exposure to both trichothecenes together induced more consistent inflammatory responses (
      • Alassane-Kpembi I.
      • Schatzmayr G.
      • Taranu I.
      • Marin D.
      • Puel O.
      • Oswald I.P.
      Mycotoxins co-contamination. Methodological aspects and biological relevance of combined toxicity studies..
      ). This explant model is perhaps the most biologically relevant ex vivo system for assessing mycotoxin effects on the intestinal epithelium because it retains the mix of cells present in the tissue, in contrast to primary or immortalized epithelial monolayer systems. Therefore, it is entirely plausible that the trichothecene exposure during this study contributed to intestinal inflammation and disease.
      On the other hand, there is evidence that ruminal microbiota can degrade DON and some other mycotoxins (
      • Seeling K.
      • Dänicke S.
      Relevance of the Fusarium toxins deoxynivalenol and zearalenone in ruminant nutrition. A review..
      ), and it is also possible that concentrations of trichothecenes arriving at the distal intestine were far less than 1 µM. One factor in this study that may have interacted with mycotoxin exposure is the relatively low effective fiber supply, which may have influenced the ruminal microbiome and its capacity for mycotoxin degradation. However, a previous study that directly tested such an interaction found no evidence that a high-concentrate diet worsened responses to a diet with much greater DON (
      • Kinoshita A.
      • Keese C.
      • Beineke A.
      • Meyer U.
      • Starke A.
      • Sauerwein H.
      • Dänicke S.
      • Rehage J.
      Effects of Fusarium mycotoxins in rations with different concentrate proportions on serum haptoglobin and hepatocellular integrity in lactating dairy cows..
      ).
      There is little information on feed-borne mycotoxins and their effects on ruminant production in a commercial setting.
      • Driehuis F.
      • Spanjer M.C.
      • Scholten J.M.
      • te Giffel M.C.
      Occurrence of mycotoxins in feedstuffs of dairy cows and estimation of total dietary intakes..
      surveyed and sampled 24 different dairies and found a wide range of mycotoxin concentrations in feed, with most samples below concentrations usually considered to be concerning. Estimates of the daily mycotoxin intake came from surveying dairy managers, so it was hard to gauge actual mycotoxin exposure.
      • Korosteleva S.N.
      • Smith T.K.
      • Boermans H.J.
      Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows..
      ,
      • Korosteleva S.N.
      • Smith T.K.
      • Boermans H.J.
      Effects of feed naturally contaminated with Fusarium mycotoxins on metabolism and immunity of dairy cows..
      ) fed dairy cows TMR naturally contaminated with mycotoxins for extended periods of time (56 and 63 d, respectively). Neither study detected any effect of mycotoxins on production, even though they were feeding mycotoxins at a rate approximately 3 times that detected in the present study.
      • Korosteleva S.N.
      • Smith T.K.
      • Boermans H.J.
      Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows..
      noted a depression of circulating immunoglobulin A, which the authors attributed to mycotoxin suppression of the immune system. Such immunosuppression could make the cow more susceptible to other challenges.
      Blood samples collected every 2 wk throughout the study were analyzed for haptoglobin, an acute-phase protein that is elevated during systemic inflammation (
      • Jacobsen S.
      • Anderson P.H.
      • Toelboell T.
      • Heegaard P.M.H.
      Dose dependency and individual variability of the lipopolysaccharide induced bovine acute phase protein response..
      ;
      • Vels L.
      • Røntved C.M.
      • Bjerring M.
      • Ingvartsen K.L.
      Cytokine and acute phase protein gene expression in repeated liver biopsies of dairy cows with a lipopolysaccharide-induced mastitis..
      ). Plasma haptoglobin concentrations (Figure 5A) tended to be greater in second-lactation cows versus parity 3+ cows (P = 0.08), and wk 1 differed from wk 3, 5, 9, 11, and 13 (P < 0.05). The temporal pattern aligned with visual observations of digestive dysfunction (e.g., excessive gut motility, loose fecal consistency) and suggested that changes implemented in wk 7 to 8 likely had a positive effect on the inflammatory status of cows by wk 11. Previous research has established that haptoglobin increases with heat stress in ruminants (
      • Alberghina D.
      • Piccione G.
      • Casella S.
      • Panzera M.
      • Morgante M.
      • Gianesella M.
      The effect of the season on some blood metabolites and haptoglobin in dairy cows during postpartum period..
      ;
      • Hamzaoui S.
      • Salama A.A.K.
      • Albanell E.
      • Such X.
      • Caja G.
      Physiological responses and lactational performances of late-lactation dairy goats under heat stress conditions..
      ;
      • Leiva T.
      • Cooke R.F.
      • Brandāo A.P.
      • Schubach K.M.
      • Batista L.F.D.
      • Miranda M.F.
      • Colombo E.A.
      • Rodrigues R.O.
      • Junior J.R.G.
      • Cerri R.L.A.
      • Vasconcelos J.L.M.
      Supplementing an immunomodulatory feed ingredient to modulate thermoregulation, physiologic, and production responses in lactating dairy cows under heat stress conditions..
      ). However, trichothecene mycotoxins have not altered haptoglobin concentrations in previous studies in lactating dairy cattle (
      • Korosteleva S.N.
      • Smith T.K.
      • Boermans H.J.
      Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows..
      ,
      • Korosteleva S.N.
      • Smith T.K.
      • Boermans H.J.
      Effects of feed naturally contaminated with Fusarium mycotoxins on metabolism and immunity of dairy cows..
      ). Furthermore, haptoglobin results varied in swine fed mycotoxins (
      • Swamy H.V.L.N.
      • Smith T.K.
      • MacDonald E.J.
      • Karrow N.A.
      • Woodward B.
      • Boermans H.J.
      Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on growth and immunological measurements of starter pigs, and the efficacy of a polymeric glucomannan mycotoxin adsorbent..
      ;
      • Díaz-Llano G.
      • Smith T.K.
      The effects of feeding grains naturally contaminated with Fusarium mycotoxins with and without a polymeric glucomannan adsorbent on lactation, serum chemistry, and reproductive performance after weaning of first-parity lactating sows..
      ;
      • Wu L.
      • Liao P.
      • He L.
      • Ren W.
      • Yin J.
      • Duan J.
      • Li T.
      Growth performance, serum biochemical profile, jejunal morphology, and the expression of nutrients transporter genes in deoxynivalenol (DON)- challenged growing pigs..
      ).
      • Wu L.
      • Liao P.
      • He L.
      • Ren W.
      • Yin J.
      • Duan J.
      • Li T.
      Growth performance, serum biochemical profile, jejunal morphology, and the expression of nutrients transporter genes in deoxynivalenol (DON)- challenged growing pigs..
      reported an increase in serum haptoglobin concentrations as concentration of mycotoxins increased in diets fed to growing pigs. However,
      • Díaz-Llano G.
      • Smith T.K.
      The effects of feeding grains naturally contaminated with Fusarium mycotoxins with and without a polymeric glucomannan adsorbent on lactation, serum chemistry, and reproductive performance after weaning of first-parity lactating sows..
      reported no difference in haptoglobin concentrations of first-parity sows fed mycotoxin-infected feed compared with negative controls. As previously discussed, it is possible that the mycotoxins made the cows in the present study more susceptible to heat stress and other forms of stress, potentially exacerbating inflammatory responses.
      Figure 5
      Figure 5Plasma biomarker concentrations of cows through a spontaneous digestive disease outbreak. (A) Haptoglobin; (B) d-lactate; (C) diamine oxidase; (D) lipopolysaccharide binding protein (LBP); and (E) serum amyloid A (SAA) among cows in parity 2 or 3+. Values are means ± SE. #P < 0.05 versus wk 1.
      d-lactate concentrations (Figure 5B) in plasma tended (P = 0.051) to be greater in second-lactation cows compared with parity 3+ cows, and wk 7 and 13 differed from wk 1 (P < 0.05). d-lactate is produced primarily by microbial metabolism; its increased concentration in plasma by wk 7 likely indicates a decline in gut barrier function (leaky gut) or excessive hindgut fermentation, with an apparent return to a more normal status by the end of the study.
      • Harmon D.L.
      • Britton R.A.
      • Prior R.L.
      • Stock R.A.
      Net portal absorption of lactate and volatile fatty acids in steers experiencing glucose-induced acidosis or fed 70% concentrate diet ad libitum..
      found increased blood d-lactate concentration in steers with induced acute acidosis compared with negative controls. Elevated d-lactate blood concentrations have been associated with acidosis and clinical signs of illness (diarrhea, behavior, eating habits, and so on) in calves (
      • Lorenz I.
      Investigations on the influence of serum D-lactate levels on clinical signs in calves with metabolic acidosis..
      ). Previous research has concluded there is a greater correlation between hindgut d-lactate production and serum d-lactate concentrations than d-lactate produced in the rumen (

      Grude, T., I. Lorenz, G. Rademacher, A. Gentile, and W. Klee. 1999. Levels of D- and L-lactate in rumen liquid, blood, and urine in calves with and without evidence of ruminal drinking. Pages 213–214 in Proc. Thirty-Second Annu. Conf. Am. Assoc. Bovine Pract. Am. Assoc. Bovine Pract.

      ;
      • Ewaschuk J.B.
      • Naylor J.M.
      • Palmer R.
      • Whiting S.J.
      • Zello G.A.
      D-lactate production and excretion in diarrheic calves..
      ;
      • Lorenz I.
      Investigations on the influence of serum D-lactate levels on clinical signs in calves with metabolic acidosis..
      ). In the first 5 wk of the study, there were significant decreases in milk fat concentrations (Figure 2C), which likely reflects some of the same changes to the microbial ecosystem that drove increased d-lactate production.
      Diamine oxidase is a digestive enzyme produced by epithelial cells in the intestine, and lysed enterocytes are thought to be the primary source of DAO in the blood stream (
      • Wollin A.
      • Wang X.
      • Tso P.
      Nutrients regulate diamine oxidase release from intestinal mucosa..
      ). Plasma DAO was therefore evaluated as a potential proxy for intestinal enterocyte mass. Diamine oxidase activity (Figure 5C) was greater in parity 2 versus parity 3+ cows (P = 0.01). Plasma DAO activity was decreased in wk 5 to 13 compared with wk 1 (all P < 0.05), independent of parity. As a relatively unexplored biomarker in plasma, interpretation of the DAO data is not straightforward (
      • Celi P.
      • Verlhac V.
      • Pérez Calvo E.
      • Schmeisser J.
      • Kluenter A.-M.
      Biomarkers of gastrointestinal functionality in animal nutrition and health..
      ); the concentration of the enzyme is a function of both enterocyte mass and the rate of enterocyte lysis. In our findings, though, plasma DAO activity was clearly reduced during the weeks when clinical signs pointed to gastrointestinal distress in these cows, coincident with markers of dysbiosis (d-lactate) and systemic inflammation (haptoglobin). In alignment with our findings,
      • Fukuda T.
      • Tsukano K.
      • Nakatsuji H.
      • Suzuki K.
      Plasma diamine oxidase activity decline with diarrhea severity in calves indicating systemic dysfunction related to intestinal mucosal damage..
      reported that plasma DAO concentration (determined by ELISA, not enzymatic assay) was reduced in preruminant calves with diarrhea. These results suggest that plasma and mucosal DAO activity may both decrease during periods of intestinal disease in cattle. This suggests that release of DAO from enterocytes into the bloodstream may occur (to some degree) with normal enterocyte function and turnover, and that diminished total enterocyte mass during intestinal disease may decrease DAO appearance.
      We are not aware of published data on blood DAO activity among cattle fed mycotoxins, but pigs fed Fusarium-infected feed showed increased blood DAO (
      • Ji X.
      • Zhang Q.
      • Zheng W.
      • Yao W.
      Morphological and molecular response of small intestine to lactulose and hydrogen-rich water in female piglets fed Fusarium mycotoxins contaminated diet..
      ). Furthermore, poultry fed Fusarium-infected feed for 42 d also had increased blood DAO compared with negative controls (
      • Cheng Y.
      • Xu Q.
      • Chen Y.
      • Su Y.
      • Wen C.
      • Zhou Y.
      Modified palygorskite improves immunity, antioxidant ability, intestinal morphology, and barrier function in broiler chickens fed naturally contaminated diet with permitted feed concentrations of Fusarium mycotoxins..
      ). We speculate that compounds such as mycotoxins that act as stressors—but may not lead to overt intestinal disease—promote adaptive changes in the mucosa that enhance DAO activity in the tissue, and in turn, in plasma. We observed greater plasma DAO concentrations among second-parity cows, the group that also had greater inflammatory biomarker concentrations and clinical disease, although there was no evidence of a time × parity interaction. Currently, there is limited published research on DAO as an intestinal health marker, and more research is required to validate its utility for investigating enteric health in cattle.
      Lipopolysaccharide binding protein (Figure 5D) did not differ by parity (P > 0.10), but plasma LBP concentration decreased in wk 3, then increased in wk 9 to 13 relative to wk 1 (P < 0.05). Lipopolysaccharide (LPS) is an endotoxin produced by gram-negative bacteria, which is released upon their lysis. Lipopolysaccharide binding protein binds LPS, limiting its immunogenic potency and enhancing its clearance from circulation (
      • Schumann R.R.
      • Leong S.R.
      • Flaggs G.W.
      • Gray P.W.
      • Wright S.D.
      • Mathison J.C.
      • Tobias P.S.
      • Ulevitch R.J.
      Structure and function of lipopolysaccharide binding protein..
      ;
      • Wright S.D.
      • Ramos R.A.
      • Tobias P.S.
      • Ulevitch R.J.
      • Mathison J.C.
      CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein..
      ). As an acute-phase protein, increased circulating LBP concentrations indicate that more inflammatory agents, potentially including LPS or other signals, were reaching the liver and altering its function (
      • Bradford B.J.
      • Yuan K.
      • Farney J.K.
      • Mamedova L.K.
      • Carpenter A.J.
      Invited Review: Inflammation during the transition to lactation: New adventures with an old flame..
      ). We are unable to explain the surprising differences in temporal patterns in haptoglobin versus LBP, given that both are acute-phase proteins expected to be induced by similar stimuli. Clinical observations of disrupted health certainly aligned better with the time period of haptoglobin elevation rather than LBP in this study. Recent findings suggested that the most important cellular source of LBP may be enterocytes, which release lipoproteins enriched in LBP to prevent hepatic inflammation after LPS translocation from the gut (
      • Han Y.-H.
      • Onufer E.J.
      • Huang L.-H.
      • Sprung R.W.
      • Davidson W.S.
      • Czepielewski R.S.
      • Wohltmann M.
      • Sorci-Thomas M.G.
      • Warner B.W.
      • Randolph G.J.
      Enterically derived high-density lipoprotein restrains liver injury through the portal vein..
      ). Considering this new information, peripheral concentrations of LBP may be less insightful than portal concentrations with respect to adaptive release of this acute-phase protein.
      Serum amyloid A is another acute-phase protein that increases in concentration when an animal is experiencing an inflammatory response. We did not detect parity or time effects for SAA (P > 0.10). The lack of significant effects for SAA is interesting considering the results for other acute-phase proteins, haptoglobin and LBP. Previous research has shown variation in SAA concentrations during a mycotoxin challenge.
      • Pate R.T.
      • Cardoso F.C.
      Injectable trace minerals (selenium, copper, zinc, and manganese) alleviate inflammation and oxidative stress during aflatoxin challenge in lactating multiparous Holstein cows..
      found no differences in SAA concentrations between lactating cows fed a diet infected with aflatoxin versus a negative control. A similar study (
      • Pate R.T.
      • Paulus Compart D.M.
      • Cardoso F.C.
      Aluminosilicate clay improves production responses and reduces inflammation during an aflatoxin challenge in lactating Holstein cows..
      ) found no differences in SAA concentrations in lactating dairy cows fed aflatoxin-infected feed compared with negative controls. Both of these studies evaluated only aflatoxins, whereas most of the mycotoxins in the present study were trichothecenes. There is little controlled research on trichothecenes and how they affect dairy cows. Swine (n = 41) fed either a diet containing deoxynivalenol or a negative control diet for 4 wk showed no increase in SAA concentrations (
      • Dänicke S.
      • Bannert E.
      • Tesch T.
      • Kersten S.
      • Frahm J.
      • Bühler S.
      • Sauerwein H.
      • Görs S.
      • Kahlert S.
      • Rothkötter H.
      • Metges C.C.
      • Kluess J.
      Oral exposure of pigs to the mycotoxins deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction..
      ). According to our findings and previous research, mycotoxins may not affect SAA concentrations, or responses may be too transient to reliably detect when sampling blood once every 2 wk.
      The drought-stressed dry corn silage had clostridia concentrations in wk 14 almost 50 times greater than samples collected in wk 9, and the concentration of clostridia in the TMR increased between wk 9 and 14 (Table 4). In contrast, fecal samples showed decreased total clostridia as well as C. perfringens between wk 9 and 14. There was therefore great disparity between the feed and fecal-sample C. perfringens microbial concentrations (Table 4); even though feed C. perfringens concentrations numerically increased in the second half of the study, fecal C. perfringens concentrations numerically decreased. This suggests a shift in the gut microbial ecosystem independent of feed contamination. Change in diet composition or in mycotoxin-microbe-gut interactions following the incorporation of mitigation strategies could have allowed for a more robust microbial ecosystem able to prevent C. perfringens dominance (
      • La Ragione R.M.
      • Woodward M.J.
      Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens..
      ;
      • Wang H.
      • Ni X.
      • Liu L.
      • Zeng D.
      • Lai J.
      • Qing X.
      • Li G.
      • Pan K.
      • Jing B.
      Controlling of growth performance, lipid deposits and fatty acid composition of chicken meat through a probiotic, Lactobacillus johnsonii during subclinical Clostridium perfringens infection..
      ). Unfortunately, microbial samples of the gut were not collected during the study, and therefore, we cannot evaluate this hypothesis. Clostridium perfringens has been implicated in several diseases within the ruminant animal, most notably jejunal hemorrhagic bowel syndrome (
      • Elhanafy M.M.
      • French D.P.
      • Braun U.
      Understanding jejunal hemorrhage syndrome..
      ). However, previous research has shown that inoculating an animal with C. perfringens does not necessarily cause the disease (
      • Abutarbush S.M.
      • Radostits O.M.
      Jejunal hemorrhage syndrome in dairy and beef cattle: 11 cases (2001 to 2003)..
      ). For this reason, is it considered an opportunistic pathogen that takes advantage when the host animal has suppressed immunity due to another stressor. None of the animals in this group experienced jejunal hemorrhagic bowel syndrome, but there is potential that the C. perfringens in the diet was an additional stress on the animals.
      Table 4Average clostridia and Clostridium perfringens enumerated in feed and fecal samples
      ItemClostridia, cfu
      Colony forming units is an approximation of viable bacteria.
      /g
      C. perfringens, cfu/g
      Wk 9Wk 14Wk 9Wk 14
      Feed samples
       TMR1,2808,70010050
       Corn silage A44021,8502050
       Corn silage B20<10<10<10
      Fecal samples34,0005,00031,0004,100
      1 Colony forming units is an approximation of viable bacteria.
      We propose that the gastrointestinal health challenges observed during this study emerged as a result of “stacked stressors.” The diet formulation likely introduced one risk factor; although not extreme, the diet was marginal in terms of supply of physically effective fiber (
      • Humer E.
      • Petri R.M.
      • Aschenbach J.R.
      • Bradford B.J.
      • Penner G.B.
      • Tafaj M.
      • Südekum II, K.-H.
      • Zebeli Q.
      Invited Review: Practical feeding management recommendations to mitigate the risk of subacute ruminal acidosis in dairy cattle..
      ), which made disturbance of the gut microbial ecosystem more likely. Second, the consistent exposure to mycotoxins likely contributed to disruption of both the microbial populations and the gut itself (
      • Park S.-H.
      • Kim D.
      • Kim J.
      • Moon Y.
      Effects of mycotoxins on mucosal microbial infection and related pathogenesis..
      ). There is growing evidence of the effects of mycotoxins specifically on the intestinal epithelium (
      • Pinton P.
      • Oswald I.P.
      Effect of deoxynivalenol and other type B trichothecenes on the intestine: A review..
      ). These effects include cytotoxicity of enterocytes, disruption of tight junction proteins, and induction of inflammatory gene expression. This disruption of the gut epithelial barrier and the associated inflammation could help trigger population or phenotype shifts in pathobionts (
      • Chandra H.
      • Sharma K.K.
      • Tuovinen O.H.
      • Sun X.
      • Shukla P.
      Pathobionts: Mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile..
      ), potentially promoting epithelial invasion and translocation of bacterial toxins. Finally, the onset of summer heat stress seemed to tip some of the cows over the edge into clinical disease. This could be related to the shift in blood flow from the internal organs to the skin in response to heat stress, which ultimately decreases oxygen supply to the gut, promoting gastrointestinal barrier disruption (

      Koch, F., U. Thom, E. Albrecht, R. Weikard, W. Nolte, B. Kuhla, and C. Kuehn. 2019. Heat stress directly impairs gut integrity and recruits distinct immune cell populations into the bovine intestine. Proc. Natl. Acad. Sci. 116:10333 LP–10338. https://doi.org/10.1073/pnas.1820130116.

      ) and further microbial dysbiosis (
      • King S.J.
      • McCole D.F.
      Epithelial-microbial diplomacy: Escalating border tensions drive inflammation in inflammatory bowel disease..
      ).
      Assuming that the proposed etiology is correct, what could have been done to avoid these problems? Because we did not see a drop in the measurable mycotoxins (Figure 3) or culturable clostridia (Table 4) in feed during the study, and heat stress continued to challenge the cows (Figure 1), the resolution of disease biomarkers and clinical signs of digestive problems by wk 11 to 13 suggests that at least some of our changes were likely effective. Increasing dietary forage has a protective role in microbial stability in the gut (
      • Lindberg J.E.
      Fiber effects in nutrition and gut health in pigs..
      ) and directly contributes to slowing passage of feed through the gastrointestinal tract (
      • Poore M.H.
      • Moore J.A.
      • Swingle R.S.
      Differential passage rates and digestion of neutral detergent fiber from grain and forages in 30, 60, and 90% concentrate diets fed to steers..
      ). Organic acid treatment of the silage face (particularly for an excessively dry silage) can help to limit fungal growth at feed-out (
      • Kung Jr., L.
      • Robinson J.R.
      • Ranjit N.K.
      • Chen J.H.
      • Golt C.M.
      • Pesek J.D.
      Microbial populations, fermentation end-products, and aerobic stability of corn silage treated with ammonia or a propionic acid-based preservative..
      ;
      • Driehuis F.
      • Elferink S.J.W.H.O.
      • Spoelstra S.F.
      Anaerobic lactic acid degradation during ensilage of whole crop maize inoculated with Lactobacillus buchneri inhibits yeast growth and improves aerobic stability..
      ), and mycotoxin binding products are effective at binding at least some mycotoxins (
      • Vila-Donat P.
      • Marín S.
      • Sanchis V.
      • Ramos A.J.
      A review of the mycotoxin adsorbing agents with an emphasis on their multi-binding capacity, for animal feed decontamination..
      ), helping to wash them out of the gut with less effect. Unfortunately, due to variable weather patterns in the United States, mycotoxins are becoming more prevalent in animal feeding programs and need to be addressed (
      • Magan N.
      • Medina A.
      • Aldred D.
      Possible climate-change effects on mycotoxin contamination of food crops pre- and postharvest..
      ;
      • Wu F.
      • Bhatnagar D.
      • Bui-klimke T.
      • Carbone I.
      • Hellmich R.
      • Munkvold G.
      • Paul P.
      • Payne G.
      • Takle E.
      Climate change impacts on mycotoxin risks in US maize..
      ;
      • Hendel E.
      • Ramirez S.
      • Gott P.
      • Raj Murugesan G.
      • Hofstetter U.
      Trends in mycotoxin contamination in United States corn..
      ).

      APPLICATIONS

      Few publications have reported temporal responses of lactating dairy cows through a spontaneous gastrointestinal health challenge. We found that intestinal disease coincided with increased plasma concentrations of the acute-phase protein haptoglobin as well as the microbial metabolite d-lactate, whereas the enterocyte-derived enzyme diamine oxidase was decreased in plasma. We further observed decreased secretion of preformed fatty acids in milk and reduced feed efficiency. A combination of suboptimal diet quality issues (mycotoxins, pathogen load, and limited physically effective fiber), compounded with seasonal heat stress, likely encouraged growth of opportunistic pathogens in the hindgut and altered gastrointestinal function. We hope that these observations can inform the development of testable hypotheses that may lead to a more robust understanding of how stacked stressors promote gastrointestinal disease.

      ACKNOWLEDGMENTS

      The authors thank Trouw Nutrition (Amersfoort, the Netherlands) for partial funding of this research.

      LITERATURE CITED

        • Abutarbush S.M.
        • Radostits O.M.
        Jejunal hemorrhage syndrome in dairy and beef cattle: 11 cases (2001 to 2003)..
        Can. Vet. J. 2005; 46: 711-715
        • Alassane-Kpembi I.
        • Schatzmayr G.
        • Taranu I.
        • Marin D.
        • Puel O.
        • Oswald I.P.
        Mycotoxins co-contamination. Methodological aspects and biological relevance of combined toxicity studies..
        Crit. Rev. Food Sci. Nutr. 2017; 57: 3489-3507
        • Alberghina D.
        • Piccione G.
        • Casella S.
        • Panzera M.
        • Morgante M.
        • Gianesella M.
        The effect of the season on some blood metabolites and haptoglobin in dairy cows during postpartum period..
        Arch. Tierzucht. 2013; 56: 354-359
        • Allen J.D.
        • Hall L.W.
        • Collier R.J.
        • Smith J.F.
        Effect of core body temperature, time of day, and climate conditions on behavioral patterns of lactating dairy cows experiencing mild to moderate heat stress..
        J. Dairy Sci. 2015; 98: 118-127
        • Bannerman D.D.
        • Paape M.J.
        • Hare W.R.
        • Sohn E.J.
        Increased levels of LPS-binding protein in bovine blood and milk following bacterial lipopolysaccharide challenge..
        J. Dairy Sci. 2003; 86: 3128-3137
        • Bradford B.J.
        • Yuan K.
        • Farney J.K.
        • Mamedova L.K.
        • Carpenter A.J.
        Invited Review: Inflammation during the transition to lactation: New adventures with an old flame..
        J. Dairy Sci. 2015; 98: 6631-6650
        • Celi P.
        • Verlhac V.
        • Pérez Calvo E.
        • Schmeisser J.
        • Kluenter A.-M.
        Biomarkers of gastrointestinal functionality in animal nutrition and health..
        Anim. Feed Sci. Technol. 2019; 250: 9-31
        • Chandra H.
        • Sharma K.K.
        • Tuovinen O.H.
        • Sun X.
        • Shukla P.
        Pathobionts: Mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile..
        Gut Microbes. 2021; 13 (1979882)
        • Cheng Y.
        • Xu Q.
        • Chen Y.
        • Su Y.
        • Wen C.
        • Zhou Y.
        Modified palygorskite improves immunity, antioxidant ability, intestinal morphology, and barrier function in broiler chickens fed naturally contaminated diet with permitted feed concentrations of Fusarium mycotoxins..
        Toxins (Basel). 2018; 10: 482
        • Cooke R.F.
        • Arthington J.D.
        Concentrations of haptoglobin in bovine plasma determined by ELISA or a colorimetric method based on peroxidase activity..
        J. Anim. Physiol. Anim. Nutr. (Berl.). 2012; 97: 531-536
        • Dänicke S.
        • Bannert E.
        • Tesch T.
        • Kersten S.
        • Frahm J.
        • Bühler S.
        • Sauerwein H.
        • Görs S.
        • Kahlert S.
        • Rothkötter H.
        • Metges C.C.
        • Kluess J.
        Oral exposure of pigs to the mycotoxins deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction..
        Innate Immun. 2020; 26: 716-732
        • Dennison A.C.
        • VanMetre D.C.
        • Callan R.J.
        • Dinsmore P.
        • Mason G.L.
        • Ellis R.P.
        Hemorrhagic bowel syndrome in dairy cattle: 22 cases (1997–2000)..
        J. Am. Vet. Med. Assoc. 2002; 221: 686-689
        • DePeters E.J.
        • Smith N.E.
        • Acedo-Rico J.
        Three to two times daily milking of older cows, and first lactation cows for entire lactations..
        J. Dairy Sci. 1985; 68: 123-132
        • Díaz-Llano G.
        • Smith T.K.
        The effects of feeding grains naturally contaminated with Fusarium mycotoxins with and without a polymeric glucomannan adsorbent on lactation, serum chemistry, and reproductive performance after weaning of first-parity lactating sows..
        J. Anim. Sci. 2007; 85: 1412-1423
        • Driehuis F.
        • Elferink S.J.W.H.O.
        • Spoelstra S.F.
        Anaerobic lactic acid degradation during ensilage of whole crop maize inoculated with Lactobacillus buchneri inhibits yeast growth and improves aerobic stability..
        J. Appl. Microbiol. 2001; 87: 583-594
        • Driehuis F.
        • Spanjer M.C.
        • Scholten J.M.
        • te Giffel M.C.
        Occurrence of mycotoxins in feedstuffs of dairy cows and estimation of total dietary intakes..
        J. Dairy Sci. 2008; 91: 4261-4271
        • Elhanafy M.M.
        • French D.P.
        • Braun U.
        Understanding jejunal hemorrhage syndrome..
        J. Am. Vet. Med. Assoc. 2013; 243: 352-358
        • Ewaschuk J.B.
        • Naylor J.M.
        • Palmer R.
        • Whiting S.J.
        • Zello G.A.
        D-lactate production and excretion in diarrheic calves..
        J. Vet. Intern. Med. 2004; 18: 744-747
        • Ewoldt J.M.
        • Anderson D.E.
        Determination of the effect of single abomasal or jejunal inoculation of Clostridium perfringens type A in dairy cows..
        Can. Vet. J. 2005; 46: 821-824
        • Feijó Corrêa J.A.F.
        • Orso P.B.
        • Bordin K.
        • Hara R.V.
        • Bittencourt Luciano F.
        Toxicological effects of fumonisin B1 in combination with other Fusarium toxins..
        Food Chem. Toxicol. 2018; 121: 483-494
        • Fukuda T.
        • Tsukano K.
        • Nakatsuji H.
        • Suzuki K.
        Plasma diamine oxidase activity decline with diarrhea severity in calves indicating systemic dysfunction related to intestinal mucosal damage..
        Res. Vet. Sci. 2019; 126: 127-130
        • Godden S.
        • Frank R.
        • Ames T.
        Survey of Minnesota dairy veterinarians on the occurrence of and potential risk factors for jejunal hemorrhage syndrome in adult dairy cows..
        Bov. Pract. 2001; 35: 97-103
        • Grenier B.
        • Applegate T.J.
        Modulation of intestinal functions following mycotoxin ingestion: Meta-analysis of published experiments in animals..
        Toxins (Basel). 2013; 5: 396-430
        • Gressley T.F.
        • Davison K.A.
        • Macies J.
        • Leonardi C.
        • McCarthy M.M.
        • Nemec L.M.
        • Rice C.A.
        Effect of abomasal carbohydrates and live yeast on measures of postruminal fermentation..
        J. Anim. Sci. 2016; 94: 284-296
        • Gressley T.F.
        • Hall M.B.
        • Armentano L.E.
        Ruminant Nutrition Symposium: Productivity, digestion, and health responses to hindgut acidosis in ruminants..
        J. Anim. Sci. 2011; 89: 1120-1130
      1. Grude, T., I. Lorenz, G. Rademacher, A. Gentile, and W. Klee. 1999. Levels of D- and L-lactate in rumen liquid, blood, and urine in calves with and without evidence of ruminal drinking. Pages 213–214 in Proc. Thirty-Second Annu. Conf. Am. Assoc. Bovine Pract. Am. Assoc. Bovine Pract.

        • Hammami H.
        • Vandenplas J.
        • Vanrobays M.-L.
        • Rekik B.
        • Bastin C.
        • Gengler N.
        Genetic analysis of heat stress effects on yield traits, udder health, and fatty acids of Walloon Holstein cows..
        J. Dairy Sci. 2015; 98: 4956-4968
        • Hamzaoui S.
        • Salama A.A.K.
        • Albanell E.
        • Such X.
        • Caja G.
        Physiological responses and lactational performances of late-lactation dairy goats under heat stress conditions..
        J. Dairy Sci. 2013; 96: 6355-6365
        • Han Y.-H.
        • Onufer E.J.
        • Huang L.-H.
        • Sprung R.W.
        • Davidson W.S.
        • Czepielewski R.S.
        • Wohltmann M.
        • Sorci-Thomas M.G.
        • Warner B.W.
        • Randolph G.J.
        Enterically derived high-density lipoprotein restrains liver injury through the portal vein..
        Science. 2021; 373 (eabe6729)
        • Harmon D.L.
        • Britton R.A.
        • Prior R.L.
        • Stock R.A.
        Net portal absorption of lactate and volatile fatty acids in steers experiencing glucose-induced acidosis or fed 70% concentrate diet ad libitum..
        J. Anim. Sci. 1985; 60: 560-569
        • Hendel E.
        • Ramirez S.
        • Gott P.
        • Raj Murugesan G.
        • Hofstetter U.
        Trends in mycotoxin contamination in United States corn..
        J. Anim. Sci. 2020; 98: 172-173
        • Humer E.
        • Petri R.M.
        • Aschenbach J.R.
        • Bradford B.J.
        • Penner G.B.
        • Tafaj M.
        • Südekum II, K.-H.
        • Zebeli Q.
        Invited Review: Practical feeding management recommendations to mitigate the risk of subacute ruminal acidosis in dairy cattle..
        J. Dairy Sci. 2018; 101: 872-888
        • Jacobsen S.
        • Anderson P.H.
        • Toelboell T.
        • Heegaard P.M.H.
        Dose dependency and individual variability of the lipopolysaccharide induced bovine acute phase protein response..
        J. Dairy Sci. 2004; 87: 3330-3339
        • Ji X.
        • Zhang Q.
        • Zheng W.
        • Yao W.
        Morphological and molecular response of small intestine to lactulose and hydrogen-rich water in female piglets fed Fusarium mycotoxins contaminated diet..
        J. Anim. Sci. Biotechnol. 2019; 10: 9
        • Kadzere C.T.
        • Murphy M.R.
        • Silanikove N.
        • Maltz E.
        Heat stress in lactating dairy cows: A review..
        Livest. Prod. Sci. 2002; 77: 59-91
        • King S.J.
        • McCole D.F.
        Epithelial-microbial diplomacy: Escalating border tensions drive inflammation in inflammatory bowel disease..
        Intest. Res. 2019; 17: 177-191
        • Kinoshita A.
        • Keese C.
        • Beineke A.
        • Meyer U.
        • Starke A.
        • Sauerwein H.
        • Dänicke S.
        • Rehage J.
        Effects of Fusarium mycotoxins in rations with different concentrate proportions on serum haptoglobin and hepatocellular integrity in lactating dairy cows..
        J. Anim. Physiol. Anim. Nutr. (Berl.). 2015; 99: 887-892
        • Kirkpatrick M.
        • Timms L.
        • Kersting K.W.
        • Kinyon J.
        Case study—Jejunal hemorrhage syndrome in dairy cattle..
        Bov. Pract. 2001; 35: 104-116
      2. Koch, F., U. Thom, E. Albrecht, R. Weikard, W. Nolte, B. Kuhla, and C. Kuehn. 2019. Heat stress directly impairs gut integrity and recruits distinct immune cell populations into the bovine intestine. Proc. Natl. Acad. Sci. 116:10333 LP–10338. https://doi.org/10.1073/pnas.1820130116.

        • Korosteleva S.N.
        • Smith T.K.
        • Boermans H.J.
        Effects of feedborne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows..
        J. Dairy Sci. 2007; 90: 3867-3873
        • Korosteleva S.N.
        • Smith T.K.
        • Boermans H.J.
        Effects of feed naturally contaminated with Fusarium mycotoxins on metabolism and immunity of dairy cows..
        J. Dairy Sci. 2009; 92: 1585-1593
        • Kung Jr., L.
        • Robinson J.R.
        • Ranjit N.K.
        • Chen J.H.
        • Golt C.M.
        • Pesek J.D.
        Microbial populations, fermentation end-products, and aerobic stability of corn silage treated with ammonia or a propionic acid-based preservative..
        J. Dairy Sci. 2000; 83: 1479-1486
        • Kvidera S.K.
        • Dickson M.J.
        • Abuajamieh M.
        • Snider D.B.
        • Fernandez M.V.S.
        • Johnson J.S.
        • Keating A.F.
        • Gorden P.J.
        • Green H.B.
        • Schoenberg K.M.
        • Baumgard L.H.
        Intentionally induced intestinal barrier dysfunction causes inflammation, affects metabolism, and reduces productivity in lactating Holstein cows..
        J. Dairy Sci. 2017; 100: 4113-4127
        • La Ragione R.M.
        • Woodward M.J.
        Competitive exclusion by Bacillus subtilis spores of Salmonella enterica serotype Enteritidis and Clostridium perfringens in young chickens..
        Vet. Microbiol. 2003; 94: 245-256
        • Leiva T.
        • Cooke R.F.
        • Brandāo A.P.
        • Schubach K.M.
        • Batista L.F.D.
        • Miranda M.F.
        • Colombo E.A.
        • Rodrigues R.O.
        • Junior J.R.G.
        • Cerri R.L.A.
        • Vasconcelos J.L.M.
        Supplementing an immunomodulatory feed ingredient to modulate thermoregulation, physiologic, and production responses in lactating dairy cows under heat stress conditions..
        J. Dairy Sci. 2017; 100: 4829-4838
        • Li C.
        • Beauchemin K.A.
        • Yang W.
        Feeding diets varying in forage proportion and particle length to lactating dairy cows: I. Effects on ruminal pH and fermentation, microbial protein synthesis, digestibility, and milk production..
        J. Dairy Sci. 2020; 103: 4340-4354
        • Lindberg J.E.
        Fiber effects in nutrition and gut health in pigs..
        J. Anim. Sci. Biotechnol. 2014; 5: 15
        • Lorenz I.
        Investigations on the influence of serum D-lactate levels on clinical signs in calves with metabolic acidosis..
        Vet. J. 2004; 168: 323-327
        • Magan N.
        • Medina A.
        • Aldred D.
        Possible climate-change effects on mycotoxin contamination of food crops pre- and postharvest..
        Plant Pathol. 2011; 60: 150-163
        • Ominski K.H.
        • Kennedy A.D.
        • Wittenberg K.M.
        • Moshtaghi Nia S.A.
        Physiological and production responses to feeding schedule in lactating dairy cows exposed to short-term, moderate heat stress..
        J. Dairy Sci. 2002; 85: 730-737
        • Owaki S.
        • Kawabuchi S.
        • Ikemitsu K.
        • Shono H.
        • Furuoka H.
        Pathological findings of hemorrhagic bowel syndrome (HBS) in six dairy cattle cases..
        J. Vet. Med. Sci. 2015; 77: 879-881
        • Park S.-H.
        • Kim D.
        • Kim J.
        • Moon Y.
        Effects of mycotoxins on mucosal microbial infection and related pathogenesis..
        Toxins (Basel). 2015; 7: 4484-4502
        • Pate R.T.
        • Cardoso F.C.
        Injectable trace minerals (selenium, copper, zinc, and manganese) alleviate inflammation and oxidative stress during aflatoxin challenge in lactating multiparous Holstein cows..
        J. Dairy Sci. 2018; 101: 8532-8543
        • Pate R.T.
        • Paulus Compart D.M.
        • Cardoso F.C.
        Aluminosilicate clay improves production responses and reduces inflammation during an aflatoxin challenge in lactating Holstein cows..
        J. Dairy Sci. 2018; 101: 11421-11434
        • Pinton P.
        • Oswald I.P.
        Effect of deoxynivalenol and other type B trichothecenes on the intestine: A review..
        Toxins (Basel). 2014; 6: 1615-1643
        • Polsky L.
        • von Keyserlingk M.A.G.
        Invited review: Effects of heat stress on dairy cattle welfare..
        J. Dairy Sci. 2017; 100: 8645-8657
        • Poore M.H.
        • Moore J.A.
        • Swingle R.S.
        Differential passage rates and digestion of neutral detergent fiber from grain and forages in 30, 60, and 90% concentrate diets fed to steers..
        J. Anim. Sci. 1990; 68: 2965-2973
        • Sanchez J.P.
        • Misztal I.
        • Aguilar I.
        • Zumbach B.
        • Rekaya R.
        Genetic determination of the onset of heat stress on daily milk production in the US Holstein cattle..
        J. Dairy Sci. 2009; 92: 4035-4045
        • Schumann R.R.
        • Leong S.R.
        • Flaggs G.W.
        • Gray P.W.
        • Wright S.D.
        • Mathison J.C.
        • Tobias P.S.
        • Ulevitch R.J.
        Structure and function of lipopolysaccharide binding protein..
        Science. 1990; 249: 1429-1431
        • Seeling K.
        • Dänicke S.
        Relevance of the Fusarium toxins deoxynivalenol and zearalenone in ruminant nutrition. A review..
        J. Anim. Feed Sci. 2005; 14: 3-40
        • St. Pierre N.R.
        • Cobanov B.
        • Schnitkey G.
        Economic losses from heat stress by US livestock industries..
        J. Dairy Sci. 2003; 86: 52-77
        • Swamy H.V.L.N.
        • Smith T.K.
        • MacDonald E.J.
        • Karrow N.A.
        • Woodward B.
        • Boermans H.J.
        Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on growth and immunological measurements of starter pigs, and the efficacy of a polymeric glucomannan mycotoxin adsorbent..
        J. Anim. Sci. 2003; 81: 2792-2803
        • Vels L.
        • Røntved C.M.
        • Bjerring M.
        • Ingvartsen K.L.
        Cytokine and acute phase protein gene expression in repeated liver biopsies of dairy cows with a lipopolysaccharide-induced mastitis..
        J. Dairy Sci. 2009; 92: 922-934
        • Vila-Donat P.
        • Marín S.
        • Sanchis V.
        • Ramos A.J.
        A review of the mycotoxin adsorbing agents with an emphasis on their multi-binding capacity, for animal feed decontamination..
        Food Chem. Toxicol. 2018; 114: 246-259
        • Wang H.
        • Ni X.
        • Liu L.
        • Zeng D.
        • Lai J.
        • Qing X.
        • Li G.
        • Pan K.
        • Jing B.
        Controlling of growth performance, lipid deposits and fatty acid composition of chicken meat through a probiotic, Lactobacillus johnsonii during subclinical Clostridium perfringens infection..
        Lipids Health Dis. 2017; 16: 38
        • West J.W.
        Effects of heat-stress on production in dairy cattle..
        J. Dairy Sci. 2003; 86: 2131-2144
        • Wollin A.
        • Wang X.
        • Tso P.
        Nutrients regulate diamine oxidase release from intestinal mucosa..
        Am. J. Physiol. Regul. Integr. Comp. Physiol. 1998; 275: R969-R975
        • Wright S.D.
        • Ramos R.A.
        • Tobias P.S.
        • Ulevitch R.J.
        • Mathison J.C.
        CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein..
        Science. 1990; 249: 1431-1433
        • Wu F.
        • Bhatnagar D.
        • Bui-klimke T.
        • Carbone I.
        • Hellmich R.
        • Munkvold G.
        • Paul P.
        • Payne G.
        • Takle E.
        Climate change impacts on mycotoxin risks in US maize..
        World Mycotoxin J. 2011; 4: 79-93
        • Wu L.
        • Liao P.
        • He L.
        • Ren W.
        • Yin J.
        • Duan J.
        • Li T.
        Growth performance, serum biochemical profile, jejunal morphology, and the expression of nutrients transporter genes in deoxynivalenol (DON)- challenged growing pigs..
        BMC Vet. Res. 2015; 11: 144
        • Yang L.
        • Tu D.
        • Wu Y.
        • Liu Y.
        • Hu Y.
        • Liu T.
        • Tan L.
        • Li Y.
        • Lei H.
        • Zhan Y.
        • Wang N.
        • Deng Z.
        • Guo S.
        • Wang A.
        Distribution and persistence of residual T-2 and HT-2 toxins from moldy feed in broiler chickens..
        Toxicon. 2020; 178: 82-91
        • Zimbleman R.B.
        • Rhoads R.P.
        • Baumgard L.H.
        • Collier R.J.
        Revised temperature humidity index (THI) for high producing dairy cows..
        J. Dairy Sci. 2009; 92: 347