Evaluation of fecal smear methods for research on Cryptosporidium spp. oocysts in the feces of dairy calves

The objective of this study is to compare the direct fecal smear (DFS) and centrifugal sedimentation (CS) methods in the detection of Cryptosporidium spp. oocysts in fecal samples of dairy calves. One hundred and fourteen fecal samples were collected from calves aged up to six months from 10 dairy farms located in Palotina and Francisco Alves, Paraná, Brazil. The microscopic analysis revealed the presence of Cryptosporidium spp. oocysts in 51.75% (59/114) of the samples in both methods. In CS, 48.25% (55/114) of the samples were positive, while in DFS slides, only 6.14% (7/114) were positive. Only 4 samples were positive exclusively in DFS. To ensure that there were no false-negative results in the microscopic analysis, the 55 samples that were negative in both DFS and CS were selected for molecular analysis using the nested PCR (nPCR). Of these 55 samples, 24% (13/55) were positive and forwarded for sequencing part of the genome, which made it possible to identify C. parvum, C. bovis and C. ryanae. Besides the characterization of the Cryptosporidium species, it was possible to identify bacteria of the genus Acinetobacter interfering directly in the analyzed samples. The microscopic analysis also revealed higher sensitivity when CS was used to make the fecal smears. However, some samples that were negative in this technique had positive PCR results. Thus, molecular analysis is indicated to confirm cases of Cryptosporidium spp. Further studies are necessary to prove the specificities of the used primers since the results obtained in nPCR were positive for the protozoan but, when genetic sequencing was performed, Acinetobacter spp. was identified.

Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

Deltamethrin Application on Pre-Weaned Calves Improves Feed Consumption, Stress and Fatigue Status under Heat Stress Conditions

Fly infestation remains a universal problem for dairy cattle herds, affecting the animals’ health and welfare status. Pre-weaned dairy calves are significantly challenged by the direct and indirect consequences of severe fly infestation, heat-stress and their interaction, which contribute to a stressful and fatiguing environment. Among several physiological, behavioral, clinical and biochemical traits, serum cortisol (SC) and creatine kinase (CK) levels, as well as feed consumption can be used as valid indicators of potential stressful and fatiguing conditions and, therefore, can be efficiently used for stress analysis studies. Hence, the objective of the study was to assess the fly-repellency effect of deltamethrin on pre-weaned dairy calves exposed to heat stress conditions, as well as its association with SC, CK concentrations and feed consumption. Two commercial dairy cattle herds of the Holstein breed in Central Macedonia (Greece) were involved in the study during summer months and under heat stress conditions. Deltamethrin administration resulted in (i) a decreased fly population (100% Musca domestica) landing on pre-weaned dairy calves, (ii) a reduced SC (stress indicator) and CK (fatigue indicator) concentration, and (iii) an increased consumption of feedstuff in deltamethrin treated animals compared to the untreated ones.

Associations between Feeding Behaviors Collected from an Automated Milk Feeder and Neonatal Calf Diarrhea in Group Housed Dairy Calves: A Case-Control Study

The objective of this case-control study was to determine if feeding behavior data collected from an automated milk feeder (AMF) could be used to predict neonatal calf diarrhea (NCD) in the days surrounding diagnosis in pre-weaned group housed dairy calves. Data were collected from two research farms in Ontario between 2017 and 2020 where calves fed using an AMF were health scored daily and feeding behavior data (milk intake (mL/d), drinking speed (mL/min), number of rewarded or unrewarded visits) was collected. Calves with NCD were pair matched to healthy controls (31 pairs) by farm, gender, and age at case diagnosis to assess for differences in feeding behavior between case and control calves. Calves were first diagnosed with NCD on day 0, and a NCD case was defined as calves with a fecal score of ≥2 for 2 consecutive days, where control calves remained healthy. Repeated measure mixed linear regression models were used to determine if there were differences between case and control calves in their daily AMF feeding behavior data in the days surrounding diagnosis of NCD (−3 to +5 days). Calves with NCD consumed less milk on day 0, day 1, day 3, day 4 and day 5 following diagnosis compared to control calves. Calves with NCD also had fewer rewarded visits to the AMF on day −1, and day 0 compared to control calves. However, while there was a NCD status x day interaction for unrewarded visits, there was only a tendency for differences between NCD and control calves on day 0. In this study, feeding behaviors were not clinically useful to make diagnosis of NCD due to insufficient diagnostic ability. However, feeding behaviors are a useful screening tool for producers to identify calves requiring further attention.

Influence of Sodium Humate on the Growth Performance, Diarrhea Incidence, Blood Parameters, and Fecal Microflora of Pre-Weaned Dairy Calves

This study aimed to evaluate the effects of the administration of sodium humate (NaH) on the growth performance, diarrhea incidence, and fecal microflora of pre-weaned Holstein calves. In a 53-day experiment, forty healthy newborn female calves were randomly allocated to the following four treatment groups: (1) control (basal diet); (2) 1-gram NaH (basal diet extra orally supplemented with 1 g of NaH dissolved in 100 mL of milk or milk replacer daily); (3) 3-gram NaH (basal diet extra orally supplemented with 3 g of NaH dissolved in 100 mL of milk or milk replacer daily); and (4) 5-gram NaH (basal diet extra orally supplemented with 5 g of NaH dissolved in 100 mL of milk or milk replacer daily). NaH was mixed with milk (d 2–20) or milk replacer (d 21–53). Calves in the 5-gram NaH group had a higher ADG during d 1 to 21 and d 21 to 53 than the other groups did (p < 0.05). Fecal scores and diarrheal incidence were significantly lower in the 3-gram and 5-gram NaH groups than the 1-gram NaH and control groups during d 1 to 20 (p < 0.05). The serum IgA, IgG and IL-4 concentrations, and T-SOD and T-AOC activities were higher, and the serum IL-6, TNF-α, D-lactic acid, and MDA concentrations were lower in the 5-gram NaH group than the control group (p < 0.05). Furthermore, NaH supplementation increased the abundances of Bifidobacterium and Lactobacillus but decreased the abundance of Escherichia coli in feces (p < 0.05). These encouraging findings indicated that supplementation with 5 g of NaH effectively improved the immune status, antioxidant capacity, and intestinal beneficial bacteria, and further improved the growth performance and reduced the diarrhea incidence of the pre-weaned dairy calves.

The rumen liquid metatranscriptome of post-weaned dairy calves differed by pre-weaning ruminal administration of differentially-enriched, rumen-derived inocula

Background Targeted modification of the dairy calf ruminal microbiome has been attempted through rumen fluid inoculation to alter productive phenotypes later in life. However, sustainable effects of the early life interventions have not been well studied, particularly on the metabolically active rumen microbiota and its functions. This study investigated the sustained effects of adult-derived rumen fluid inoculations in pre-weaning dairy calves on the active ruminal microbiome of post-weaned dairy calves analyzed via RNA-sequencing. Results Two different adult-derived microbial inocula (bacterial- or protozoal-enriched rumen fluid; BE or PE, respectively) were administered in pre-weaned calves (3–6 weeks) followed by analyzing active rumen microbiome of post-weaned calves (9 weeks). The shared bacterial community at the genus level of 16S amplicon-seq and RNA-seq datasets was significantly different (P = 0.024), 21 out of 31 shared major bacterial genera differed in their relative abundance between the two analytic pipelines. No significant differences were found in any of the prokaryotic alpha- and beta-diversity measurements (P > 0.05), except the archaeota that differed for BE based on the Bray–Curtis dissimilarity matrix (P = 0.009). Even though the relative abundances of potentially transferred microbial and functional features from the inocula were minor, differentially abundant prokaryotic genera significantly correlated to various fermentation and animal measurements including butyrate proportion, body weight, and papillae length and counts. The overall microbial functions were affected quantitatively by BE and qualitatively by PE (P < 0.05), and this might be supported by the individual KEGG module and CAZymes profile differences. Exclusive networks between major active microbial (bacterial and archaeal genera) and functional features (KEGG modules) were determined which were differed by microbial inoculations. Conclusions This study demonstrated that actively transcribed microbial and functional features showed reliable connections with different fermentations and animal development responses through adult rumen fluid inoculations compared to our previous 16S amplicon sequencing results. Exclusive microbial and functional networks of the active rumen microbiome of dairy calves created by BE and PE might also be responsible for the different ruminal and animal characteristics. Further understanding of the other parts of the gastrointestinal tract (e.g., abomasum, omasum, and small intestine) using metatranscriptomics will be necessary to elucidate undetermined biological factors affected by microbial inoculations.

Fecal microbiome profiles of neonatal dairy calves with varying severities of gastrointestinal disease

Gastrointestinal disease (GI) is the most common illness in pre-weaned dairy calves. Studies have associated the fecal microbiome composition with health status, but it remains unclear how the microbiome changes across different levels of GI disease and breeds. Our objective was to associate the clinical symptoms of GI disease with the fecal microbiome. Fecal samples were collected from calves (n = 167) of different breeds (Holstein, Jersey, Jersey-cross and beef-cross) from 4–21 d of age. Daily clinical evaluations assessed health status. Calves with loose or watery feces were diagnosed with diarrhea and classified as bright-sick (BS) or depressed-sick (DS) according to behavior. Calves with normal or semiformed feces and no clinical illness were classified as healthy (H). One hundred and three fecal samples were obtained from consistently healthy calves and 64 samples were from calves with diarrhea (n = 39 BS; n = 25 DS). The V3-V4 region of 16S rRNA gene was sequenced and analyzed. Differences were identified by a linear-mixed effects model with a negative binomial error. DS and Jersey calves had a higher relative abundance of Streptococcus gallolyticus relative to H Holstein calves. In addition, DS calves had a lower relative abundance of Bifidobacterium longum and an enrichment of Escherichia coli. Species of the genus Lactobacillus, such as an unclassified Lactobacillus, Lactobacillus reuteri, and Lactobacillus salivarius were enriched in calves with GI disease. Moreover, we created a model to predict GI disease based on the fecal microbiome composition. The presence of Eggerthella lenta, Bifidobacterium longum, and Collinsella aerofaciens were associated with a healthy clinical outcome. Although lactobacilli are often associated with beneficial probiotic properties, the presence of E. coli and Lactobacillus species had the highest coefficients positively associated with GI disease prediction. Our results indicate that there are differences in the fecal microbiome of calves associated with GI disease severity and breed specificities.