Frontiers | Influence of Cryptosporidium and rotavirus co-infection on infectivity in calves

Frontiers | Influence of Cryptosporidium and rotavirus co-infection on infectivity in calves
ORIGINAL RESEARCH article
Front. Vet. Sci.
, 17 February 2026
Sec. Parasitology
Volume 13 - 2026 |
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ORIGINAL RESEARCH article
Front. Vet. Sci.
, 17 February 2026
Sec. Parasitology
Volume 13 - 2026 |
Influence of
Cryptosporidium
and rotavirus co-infection on infectivity in calves
Fumi Murakoshi
1,2,3
Megumi Itoh
Rofaida Mostafa Soliman
5,6
Tatsunori Masatani
7,8
Kenichi Shibano
Takaaki Nakaya
Kentaro Kato
1.
Laboratory of Veterinary Microbiology, Tokyo University of Agriculture and Technology, Tokyo, Japan
2.
Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan
3.
Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University, Osaki, Miyagi, Japan
4.
Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan
5.
Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto, Japan
6.
Department of Infectious Diseases and Epidemics, Faculty of Veterinary Medicine, Damanhour University, Damanhour, El-Beheira, Egypt
7.
Laboratory of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
8.
Center for One Medicine Innovative Translational Research (COMIT), Institute for Advanced Study, Gifu University, Gifu, Japan
9.
Faculty of Veterinary Medicine, Okayama University of Science, Okayama, Japan
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Abstract
Rotavirus A (RVA; species
Rotavirus alphagastroenteritidis
) and
Cryptosporidium
spp. are major enteric pathogens in infants and neonatal calves, causing severe diarrhea that can lead to fatal outcomes. These pathogens thus pose challenges in both public health and the livestock industries. Although co-infections are common, their pathogenesis remains poorly understood. Here, we conducted a longitudinal investigation in naturally infected calves to assess the impact of co-infection with rotavirus and
Cryptosporidium
. Infection status was determined based on daily fecal antigen testing and oocyst per gram (OPG) counts from birth to 22 days of age. Based on these criteria, seven calves were classified as having
Cryptosporidium
mono-infection and three calves as having mixed infection. We found that subclinical infection with bovine rotavirus significantly shortened the duration of diarrhea caused by
Cryptosporidium parvum
in calves and reduced initial oocyst shedding. Furthermore,
in vitro
experiments using the bovine intestinal epitheliocyte (BIE) cell line demonstrated that the BRV Lincoln strain (G6, P[1]) non-structural protein 4 (NSP4) inhibits
C. parvum
infection, possibly by interfering with the host sodium-glucose co-transporter 1 (SGLT1). Our study highlights a potential novel strategy for controlling both BRV and
C. parvum
by exploiting their interactions during co-infection.
1 Introduction
Diarrheal diseases are a major cause of morbidity and mortality in both humans and livestock worldwide. In the livestock industry, neonatal diarrhea in calves leads to significant economic losses due to growth retardation, increased mortality, and reduced productivity (
1–3
). Among young ruminants, the main causative agents include
bovine rotavirus
Cryptosporidium parvum
Escherichia coli
Salmonella enterica
, and
Clostridium perfringens
). Notably,
C. parvum
and bovine rotavirus A are among the leading causes of diarrhea in neonatal calves (
).
Cryptosporidium parvum
is a zoonotic protozoan parasite capable of infecting more than 150 vertebrate species, including humans (
). Upon oral ingestion, the parasite proliferates in the intestinal epithelium and releases oocysts that are highly resistant to chlorine-based disinfectants, facilitating environmental persistence. In calves, prophylactic administration of halofuginone lactate or lasalocid-NA has been shown to reduce the incidence of cryptosporidiosis (
). Recently, passive immunization strategies, such as maternal vaccination using
C. parvum
antigen–based vaccines (e.g., Bovilis Cryptium
, MSD Animal Health), have also been developed to prevent neonatal infection (
).
Rotavirus is a genus of double-stranded RNA viruses within the family
Reoviridae
and comprises at least 11 recognized species (A–D, F–L). Among these, rotavirus A (species Rotavirus alphagastroenteritidis), commonly referred to as bovine rotavirus (BRV) in cattle, is a major enteric pathogen in neonatal calves. In cattle, an inactivated vaccine is available for use in pregnant cows to enhance colostral antibodies, thereby preventing neonatal rotavirus disease (
10
). However, no effective antiviral treatment is currently available.
Co-infections with rotavirus and
C. parvum
have been reported in calves and may result from overlapping susceptibility periods:
C. parvum
commonly infects calves aged 1–21 days (
11
), whereas BRV primarily affects those aged 1–14 days. Previous studies reported inconsistent findings regarding the clinical outcomes of co-infection (
12
13
), and most lacked information on infection timing or prior exposure. Therefore, longitudinal investigations are necessary to clarify the pathophysiological interactions between these two pathogens in neonatal calves. Rotavirus non-structural protein 4 (NSP4) is a viral enterotoxin, and its biologically active regions have been identified in previous studies. Notably, the NSP4_114–135 peptide was originally described as a functional domain capable of inhibiting the Na
-D-glucose symporter (SGLT1) (
14
15
).
In this study, we conducted a longitudinal investigation to assess the impact of co-infection with rotavirus and
C. parvum
in calves. In addition, we performed
in vitro
molecular analyses to elucidate the interactions between the two pathogens during co-infection.
2 Methods
2.1 Investigation of calves with mixed infections of BRV and
C. parvum
2.1.1 Information on the calves used in the experiment
From February to March 2016, fecal samples were collected daily from 10 calves at a dairy farm in Obihiro City, Hokkaido, Japan. This farm raises Holstein and Jersey cattle, calves were fed 4–6 L of colostrum within 24 h after birth. Insufficient colostrum intake in neonatal calves is associated with an increased risk of rotavirus-induced diarrhea. At the study farm, when the Brix value of colostrum was below 22% (approximately equivalent to an IgG concentration of 50 mg/mL), 450 g of a bovine colostrum powder supplement (Headstart
, Elanco, Indiana, U.S.) is administered. Rotavirus and
Cryptosporidium
infections have been recognized as major health issues in calves at this farm. No vaccines against either pathogen were used on this farm. In addition to the samples collected from this farm, fecal samples were included from two calves used in practical training at Obihiro University of Agriculture and Veterinary Medicine in December 2016. Twelve calves were initially enrolled, but data from eleven were analyzed after one was transferred to another farm. The calves included in this study ranged in age from 1 to 22 days and were sampled consecutively. The study population consisted of nine Holstein and two Jersey calves, all of which were clinically healthy at birth. Among the Jersey calves, one belonged to the co-infection group and the other to the
C. parvum
-infected group. Individual infection days for BRV and
C. parvum
for each calf are summarized in
Supplementary Table S1
. Because the primary objective of this field study was to evaluate the effect of prior rotavirus infection on subsequent
C. parvum
–associated disease, only calves in which rotavirus infection preceded
C. parvum
infection were included in the main comparative analyses. Of the 11 calves with complete longitudinal data, 7 calves with
C. parvum
mono-infection and 3 calves with rotavirus-preceding co-infection were included in the main comparative analyses.
2.1.2 Fecal sample collection and pathogen detection
Housing conditions and fecal sample collection procedures were standardized throughout the study. All calves included in this study were housed individually in separate pens throughout the study period and were physically separated from other calves kept at the farm at the same time. This housing arrangement minimized the possibility of cross-contamination between animals. Fecal samples were collected daily directly from the rectum of each individual calf using disposable gloves. Fecal samples were collected in the morning, and their physical characteristics were recorded as diarrheic, soft, or normal. Diarrhea was defined as stools corresponding to types 6–7 on the Bristol Stool Scale (BSS). Subsequently, immunochromatographic test kits (DipFit Tetra Calf Scours; cat. no. BIO K 156, Bio-X Diagnostics S.A., Rochefort, Belgium) were used to detect infections caused by rotavirus,
Cryptosporidium
, coronavirus, and
Escherichia coli
E. coli
) K99 (F5). All tests were performed strictly according to the manufacturer’s instructions, without any modifications to the protocol. The collected fecal samples were stored at 4 °C. Each calf was monitored from birth (day 0) to day 22.
Shedding of
Cryptosporidium
oocysts was quantified by determining oocysts per gram (OPG) of feces using a standard sucrose flotation method, as described previously (
16
17
). Briefly, one gram of feces was placed into a 15-mL tube, and 14 mL of sucrose solution (density 1.2 g/mL) was added. The mixture was centrifuged at 1,300 rpm (corresponding to approximately 300 × g, depending on rotor radius) for 10 min. After centrifugation, the tube was filled with sucrose solution, and a coverslip was gently placed on top. The preparation was allowed to stand for 30 min, after which
Cryptosporidium
oocysts adhering to the coverslip were counted under a light microscope.
2.1.3 Species identification and subtyping using PCR
Genomic DNA was extracted from fecal samples (0.3–0.4 g) using the QIAamp Fast DNA Stool Mini Kit (cat. no. 51604; QIAGEN, Hilden, Germany), strictly following the manufacturer’s instructions.
Cryptosporidium
spp. were detected and subtyped by nested PCR amplification targeting a ~ 830 bp and a ~ 850 bp fragment of the small subunit (SSU) rRNA and 60-kDa glycoprotein (GP60) genes, respectively, as described previously (
18
19
).
2.2 Culture of BRV and
Cryptosporidium parvum
As host cells for BRV growth, African green monkey kidney cells (MA104 cells, RCB0994) were purchased from RIKEN BioResource Research Center and were maintained in minimum essential medium (MEM) (cat. no. 21443–15; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (cat. no. 09367–34; Nacalai Tesque, Kyoto, Japan). The bovine intestinal epitheliocyte (BIE) cell line (Passage 39) kindly provided by Dr. Hisashi Aso (Graduate school of agricultural science, Tohoku University, Japan) (
20
21
) was used for BRV and
C. parvum
inoculation studies; BIE cells were cultured in DMEM medium (cat. no. 16971–55; Nacalai Tesque, Kyoto, Japan) with 10% FBS and 100 U/mL penicillin, and 100 μg/mL streptomycin. BIE cells reproduce key morphological and functional features of bovine intestinal epithelium, including microvilli formation, expression of tight-junction proteins, and the epithelial marker cytokeratin. They also retain the ability to differentiate into M-like cells under appropriate conditions, indicating preservation of epithelial characteristics (
20
21
). Although it remains unclear whether BIE cells elicit interferon responses identical to those observed
in vivo
in bovine intestinal cells, their response pattern to rotavirus infection is comparable to that reported in other established
in vitro
rotavirus infection systems (
22
). Bovine rotavirus (BRV Lincoln strain (G6, P[1])) kindly provided by Dr. Kunitoshi Imai (Graduate School of Animal and Veterinary Sciences and Agriculture, Obihiro University of Agriculture and Veterinary Medicine, Japan) was propagated in the MA104 cell line (Passage 3) as previously described (
23
). Viral titers were determined using fluorescence focus units (FFU) as previously described (
24
). All virus stocks were stored at −80 °C until use. BRVs were activated by incubation with trypsin (final concentration 1 μg/mL; Sigma Aldrich, MO, USA) at 37 °C for 30 min prior to infection (
25
).
Cryptosporidium parvum
oocysts, strain HNJ-1 (
26
27
), were kindly provided by Dr. Makoto Matsubayashi (Graduate school of veterinary science, Osaka Prefecture University, Japan). Oocysts were maintained by passage in experimentally infected SCID mice (C.B-17/Icr-
scid
scid
Jcl) (CLEA Japan, Inc., Tokyo, Japan) and were purified from feces by using discontinuous sucrose and cesium chloride gradients as described previously (
17
).
C. parvum
oocysts less than 6 months since harvest were used in all experiments. Animal experiments were approved by the Ethical Committee of the Committee on Animal Experiments of the Kyoto Prefectural University of Medicine (M2019-226, M2020-247, M2021-272).
2.3 Co-infection of BIE cells with BRV and
C. parvum
Cryptosporidium parvum
oocysts were bleached with 10% (v/v) purelox (OyaloxCo.Ltd., Tokyo, Japan) on ice for 15 min, then washed three times with ice-cold phosphate-buffered saline (PBS) and incubated with 0.2 mM sodium taurocholate (Nacalai Tesque, Kyoto, Japan) at 37 °C for 30 min to stimulate excystation. BIE cells (5 × 10
cells/well) were seeded in 96-well plates and cultured for 24 h. BRV (100 μL) was incubated with trypsin at a final concentration of 1 μg/mL at 37 °C for 30 min. Following incubation, 900 μL of serum-free DMEM (cat. no. 16971–55; Nacalai Tesque, Kyoto, Japan) containing 1 μg/mL trypsin was added to the BRV-containing tube, and the resulting suspension was inoculated into 96-well plates at 100 μL per well (BRV, MOI = 1). Twelve hours after inoculation,
C. parvum
was excysted into sporozoites and added at a concentration of 5 × 10
oocysts/100 μL (MOI = 1). The plate was incubated at 37 °C for 1.5 h, washed with serum-free DMEM containing trypsin, and cultured until 24 h post-rotavirus inoculation. The experiment was conducted using a rotavirus inoculum corresponding to an MOI of 1, as determined based on infectivity titration in MA104 cells, which was chosen to ensure robust infection while minimizing excessive cytopathic effects.
The cells were fixed and permeabilized with ice-cold 100% methanol (cat. no. 137–01823; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 10 min and subsequently washed three times with PBS. Blocking was performed with 1% BSA (cat. no. 017–15,124, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in PBS for 30 min. The cells were then stained for 1 h with Sporo-Glo, an anti
-Cryptosporidium
polyclonal antibody (cat. no. A600Cy3-R-1X; Waterborne Environmental, Inc., Virginia, USA), according to the manufacturer’s instructions.
Infection with BRV induced a mild cytopathic effect (CPE) in BIE cells, characterized by partial cell detachment and a reduction in cell density. Quantitative image analysis revealed that the total number of adherent cells in BRV-infected wells was reduced by approximately 10–20% compared with non-infected control wells at 24 h post-inoculation. Because this reduction in viable cell numbers could affect the apparent frequency of subsequent
C. parvum
infection, the number of
C. parvum
-infected cells was normalized to the total number of viable cells in each well. Viable cell numbers were quantified using an IN Cell Analyzer 2,200 (GE Healthcare, Illinois, USA), with 42 fields acquired per well using a 20 × objective lens. The number of visible cell nuclei was determined using IN Cell Developer Toolbox software (GE Healthcare, Illinois, USA), and
C. parvum
infection rates were calculated as the proportion of infected cells relative to the total number of viable cells. BRV infection was confirmed by immunofluorescence assay (IFA). The cells were fixed with ice-cold 100% methanol for 10 min. Goat anti-BRV polyclonal antibodies (cat. no. ab20036; Abcam, Cambridge, UK) were used as primary antibodies to detect BRV antigens. Donkey anti-goat IgG conjugated with Alexa Fluor 555 (cat. no. AP180C; Thermo Fisher Scientific, MA, USA) was used as the secondary antibody. Images were captured using a Keyence BZ-X810 microscope (KEYENCE, Osaka, Japan).
Cryptosporidium
parasites were counted in 15 random fields per well using a 20 × objective lens.
2.4 Infection of poly(I:C)-transfected cells with
C. parvum
Poly(I:C) (a synthetic double-stranded RNA (dsRNA)) (42,424, Tocris Bioscience, Bristol, UK) molecule (50 ng) was transfected into BIE cells seeded in a 96-well plate by using the PEI MAX (Polysciences, Inc., PA, USA), according to the manufacturer’s instruction. Transfection of BIE cells with poly(I:C) prior to
C. parvum
infection was performed to induce the cellular response to dsRNA before infection. Six hours after the transfection, the BIE cells were infected with
C. parvum
(MOI = 1). Three hours post-infection, the cells were washed. The number of
C. parvum-
infected BIE cells was counted 24 h after transfection. In a separate well, a similar experiment was conducted, and RNA was extracted from the BIE cells 24 h after transfection. Total RNA was extracted from each sample using the SV Total RNA Isolation System (Promega, WI, USA); reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). Interferon β expression was confirmed by PCR using the following primers: BtIFN-βF (5′-CTTTCCAGGAGCTACAGCTTGC-3′) and BtIFNβ-R (5′-ACGACTGTCCAGGCACACCTG-3′). KOD FX Neo (TOYOBO, Japan) was used for PCR amplification; the amplicon length was 435 bp.
2.5 Experimental inhibition of BIE cell infection with
C. parvum
by NSP4 peptide
We commissioned GenScript to synthesize the following peptides: NSP4_114–135: DKLTTREIEQVELLKRIYDKLT, and scrambled peptide: IDTKLDLLYRKRKIQLVETETE. The scrambled peptide had a completely random amino acid sequence. Cytotoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with the Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan). Absorbance was measured at 450 nm using a TriStar LB 941 microplate reader (Berthold Technologies, Bad Wildbad, Germany). BIE cells were seeded in 96-well plates at a density of 5 × 10
cells/well and allowed to grow overnight. As previously described,
C. parvum
oocysts were excysted into sporozoites immediately before infection. BIE cells were then infected with
C. parvum
(MOI = 1) and treated separately with one of the following reagents in DMEM medium: 50 μM NSP4 peptide, 50 μM scrambled peptide, 0.05% phloridzin n-hydrate (approximately 0.3 μM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), or 10 μM nitazoxanide (TCI, Tokyo, Japan), which served as a positive control. Each condition was tested in triplicate wells, and the experiment was independently repeated three times. Three hours after infection, the cells were washed once. The peptide or drug (phloridzin or nitazoxanide) at the same concentration as used above was added to the medium after washing. Twenty-four hours after infection, the number of
C. parvum
infections was counted using the method described previously.
The cytotoxicity of the NSP4_114–135 peptide and scrambled peptide was tested across concentrations ranging from 30 to 200 μg/mL, and no significant toxicity was observed within this range. Based on these results, a concentration of 50 μg/mL was selected for subsequent experiments. Phlorizin was employed as a specific inhibitor of SGLT1, and nitazoxanide served as a positive control.
2.6 Experimental inhibition of glucose uptake in BIE cells
BIE cells seeded in a 96-well plate were infected with
C. parvum
as described above. At 50 min post-infection, 2-NBDG [2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino) (Cayman Chemical, Michigan, USA)] was added to achieve a final concentration of 200 μM. After a 30-min incubation, the cells were washed twice with 1 × PBS. Because 2NBDG is a fluorescent analog of glucose, it was used to examine whether glucose uptake is increased at sites of
C. parvum
infection. The fluorescence of 2-NBDG was then observed under a fluorescence microscope at 200 × magnification. Fluorescent areas were counted in 10 random fields of view to assess glucose uptake.
2.7 Statistical analyses
For
in vitro
experiments, three technical replicates were performed per experiment and the average value was determined. The experimental points represent an average of each three biological replicates (three independent experiments). Statistical analyses were performed using Prism7 (GraphPad Software, MA, USA). Statistical analyses were performed as follows: Mann–Whitney U test was used to compare the duration of diarrhea and oocyst shedding
Table 1,
and oocyst output was analyzed using two-way ANOVA followed by multiple comparisons (
Figure 1
). Data shown in
Figures 2
were analyzed using an unpaired
-test, while
Figures 4
were analyzed using one-way ANOVA. Differences were considered statistically significant at
< 0.05. Because of the small sample size in the field study (
= 7 and
= 3) and the uncertainty regarding the normality of the data distribution, a non-parametric Mann–Whitney U test was used for comparisons of diarrhea duration and initial oocyst shedding between mono-infected and co-infected calves. To further evaluate the robustness of the results, Welch’s
-test, which is more tolerant of unequal variances and sample sizes than Student’s t-test, was also performed, yielding the same qualitative conclusion. Therefore, the observed statistical significance was not dependent on the choice of parametric versus non-parametric testing.
Table 1
Group
Oocyst discharge period (days)
Duration of diarrhea (days)
C. parvum
infection (
= 7)
7.4*
4.4*
Mixed infection (
= 3)
9.0*
1.3*
Oocyst shedding period and diarrhea duration in calves with
C. parvum
infection and calves co-infected with BRV and
C. parvum.
< 0.04.
Figure 1
Figure 2
Figure 3
Figure 4
3 Results
3.1 Calves co-infected with BRV and
C. parvum
exhibit a significantly shorter duration of diarrhea and a reduction in initial oocyst shedding
All 11 analyzed calves were infected with
Cryptosporidium
within the first 16 days of life. Of these, four exhibited mixed infections with BRV and
Cryptosporidium
(Calves A–D; see
Supplementary Table S1
). Immunochromatographic test results were negative for coronavirus and
Escherichia coli
. All
Cryptosporidium
isolates from the calves were identified as
C. parvum
. Subtyping revealed that one calf (Calf C) had a mixed infection with subtypes IIaA15G2R1 and IIaA16G2R1, whereas the remaining calves were all infected with subtype IIaA16G3R1. Of the four co-infected calves, three acquired rotavirus before
C. parvum
, whereas one calf (Calf D) showed the reverse order. Therefore, because the aim was to assess the effect of prior rotavirus infection on subsequent
C. parvum
–associated disease, Calf D was excluded from the main comparative analyses. Consequently, the present analyses focused on seven calves with
C. parvum
mono-infection and only three calves that acquired
C. parvum
following rotavirus infection. The clinical course of Calf D is shown in
Supplementary Table S1
. This calf exhibited a diarrhea duration and oocyst shedding pattern comparable to those observed in calves with
C. parvum
mono-infection.
Table 1
shows the duration of oocyst shedding and the number of days calves experienced watery diarrhea. Diarrhea lasted significantly shorter in co-infected calves, averaging 1 day. In contrast, calves with
C. parvum
infection alone typically developed severe diarrhea concurrently with or the day after the onset of oocyst shedding, with a mean diarrhea duration of 4.4 days. However, the co-infected calves had a significantly longer duration of oocyst shedding.
Figure 1
shows the oocyst shedding patterns. On the first day of shedding, the OPG in co-infected calves was significantly lower than that in singly infected calves. The total oocyst output was not statistically significant. Interestingly, in the co-infected calves, no symptoms of diarrhea were observed during the initial rotavirus infection, indicating that a subclinical infection had occurred (
Supplementary Table S1
). This supports that rotavirus infection occurred under conditions of sufficient maternal immunity.
Figure 5
3.2 Co-infection of BIE cells with BRV and
Cryptosporidium parvum
results in a reduction in the number of
C. parvum
infections
To investigate the initial response during co-infection with BRV and
C. parvum
, we examined changes in the number of
C. parvum
infections in BIE cells (
Figures 2A
). The results revealed a significant reduction in the number of
C. parvum
infections in the group co-infected with BRV (
Figure 2C
).
3.3 The reduction in
C. parvum
infections during co-infection is unrelated to the type I interferon response induced by viral infection
To investigate the effect of type I interferon responses induced by viral infection on
C. parvum
infection in more detail, BIE cells were transfected with poly(I:C), a synthetic analog of double-stranded RNA, to strongly induce type I interferon expression (
Figure 3A
). Upon poly(I:C) transfection, an IFN-β band was detected; however, no IFN-β band was observed in the group that was infected with
C. parvum
without poly(I:C) transfection (
Figure 3B
). Consequently, even when type I interferon expression was strongly induced by transfecting poly(I:C) into BIE cells, there were no significant changes in the number of
C. parvum
infections (
Figure 3C
). These findings suggest that the reduction in
C. parvum
infections during co-infection might involve factors beyond the innate immune response of cells.
3.4 NSP4 peptide-mediated inhibition of
C. parvum
infection in BIE cells
We next examined whether a rotavirus-derived peptide could directly affect
C. parvum
infection. BIE cells were treated with the NSP4_114–135 peptide during
C. parvum
infection (
Figure 4A
). A cytotoxicity assay confirmed that the NSP4_114–135 peptide was non-toxic at the concentration used (50 μg/mL) (
Figure 4B
). Treatment with the NSP4 peptide significantly reduced the number of
C. parvum
-infected cells compared with the scrambled peptide control. Similar inhibitory effects were observed with phlorizin, whereas nitazoxanide served as a positive control (
Figure 4C
).
3.5 NSP4 peptide inhibits the uptake of fluorescent glucose analogs at
C. parvum
infection sites
Since
C. parvum
infection requires SGLT1, localized glucose uptake is thought to occur at infection sites. To investigate this, a fluorescent glucose analog was added to determine whether its uptake was inhibited in the presence of the NSP4 peptide (
Figure 5A
). The number of punctate fluorescent signals of 2-NBDG was reduced in the groups treated with the NSP4 peptide or the SGLT1 inhibitor phlorizin during
C. parvum
infection (
Figure 5B
). These findings suggest that the NSP4 peptide inhibits the function of SGLT1 (sodium–glucose co-transport), thereby impairing the ability of
C. parvum
to achieve complete invasion of host cells. However, no significant changes in the number of
C. parvum
infections were observed at 3 h post-inoculation (
Figure 5C
), indicating that the inhibitory effect of NSP4 on
C. parvum
infection likely occurs more than 3 h post-inoculation.
4 Discussion
The prepatent period of BRV is 1–2 days, whereas that of
C. parvum
is 3–6 days (
28
29
). Therefore, in farms where both pathogens are prevalent, co-infection typically occurs in the order of BRV followed by
C. parvum
. In this study, calves with such co-infections exhibited a significantly shorter duration of diarrhea.
A major limitation of the present field study is the very small number of calves in the co-infection group (
= 3). This reflects the constraints of working with naturally occurring infections, where both the timing and the sequence of pathogen exposure cannot be experimentally controlled. Consequently, although the observed shortening of diarrhea duration in calves that acquired
C. parvum
after rotavirus infection was statistically significant, this result should be interpreted with caution and should not be overgeneralized. The present data should be viewed as hypothesis-generating, providing preliminary evidence that rotavirus infection may modulate subsequent
C. parvum
-associated disease, rather than as a definitive demonstration of this effect. Larger, independent field studies will be required to confirm the robustness and generality of this finding.
Typically, rotavirus infection causes shortening, partial detachment, and destruction of intestinal villi, leading to diarrhea (
30
). However, in the present study, the calves in the co-infected group exhibited subclinical rotavirus infection, which suggests that colostrum intake was sufficient. Although the number of cases was limited to a single calf, one calf tested positive for
C. parvum
oocysts before testing positive for BRV; the duration of diarrhea in this calf was comparable to that observed in calves with mono-infections (
Supplementary Table S1
). The timing of infection likely affects disease severity (
31
32
). The reduced oocyst shedding and diarrhea observed in co-infected calves may be associated with modulation of mucosal immune responses and barrier function.
C. parvum
infection is primarily controlled by IFN-
–dependent Th1 responses, while IL-10 plays a protective role in preventing excessive intestinal inflammation. Rotavirus infection can transiently stimulate epithelial interferon signaling, particularly type III IFNs, and alter epithelial permeability through the action of NSP4 on tight junctions and sodium–glucose transporters. Rotavirus infection has been shown to increase IFN-γ and IL-10 levels in human cases of acute gastroenteritis (
33
) and to upregulate their expression
in vitro
using infected intestinal cell models (
34
). Such transient epithelial and immune activation might restrict
C. parvum
invasion and replication, while IL-10–mediated regulation could contribute to faster recovery of epithelial integrity and reduced diarrhea. Neonatal calves and human infants share several physiological characteristics that may influence susceptibility to enteric co-infections, including an immature intestinal barrier and developing mucosal immune system. However, differences in maternal antibody acquisition, environmental exposure, and microbial colonization patterns likely result in distinct infection dynamics between calves and human infants. Although these differences limit direct extrapolation, the epithelial and immune interactions identified in calves may provide useful insights into the mechanisms underlying co-infection in early human life.
Although serial quantification of both pathogens by qPCR would further strengthen the analyses of infection dynamics, this approach is technically difficult under field conditions, particularly when working with fecal samples that vary greatly in consistency and RNA recovery. In this study,
C. parvum
shedding was quantified by oocyst counts (OPG), which provide a reliable estimate of parasite burden, and rotavirus infection was monitored using an immunochromatographic test kit, which reflects the period of viral shedding in calves. We therefore consider the combination of OPG data and immunochromatographic detection to provide a reasonable approximation of infection timing and duration for both pathogens.
The observed reduction in diarrhea duration among co-infected calves should nevertheless be interpreted with caution, as factors such as age, colostrum intake, and immune status may also influence disease outcomes (
35
36
). Although detailed immunological parameters were not available for all animals, all calves received colostrum and no signs of failure of passive transfer were recorded. While these factors may contribute to some variability in clinical response, they are unlikely to fully account for the marked difference in diarrhea duration. Future studies incorporating these parameters as covariates will be valuable to further clarify the relationship between co-infection and disease severity.
A study investigating the clinical significance of pathogen combinations in acute diarrhea among children in Rwanda and Zanzibar found that while certain pathogen combinations exacerbated symptoms, the combination of rotavirus and
Cryptosporidium
did not, supporting the findings of the present study (
37
). However, the mechanism underlying the significantly prolonged shedding of
C. parvum
oocysts in co-infected calves remains unclear. One possibility is that the reduction in the initial infection burden results in an inadequate immune response against
C. parvum
, leading to delayed clearance. The reduction in early
C. parvum
infection in calves may be attributed to cytokine responses induced by rotavirus infection. Supporting this hypothesis, IFN-
has been reported to inhibit
C. parvum
infection (
38
). Thus, it is plausible that
in vivo
, BRV infection induces IFN-γ production in calves, thereby suppressing
C. parvum
infection. Further detailed studies of the immune response of calves to co-infection are needed. Since the decrease in cell count caused by BRV infection was normalized, the observed reduction in
C. parvum
infection cannot be explained solely by reduced cell availability, indicating that additional BRV-induced factors are likely involved. Previous studies suggest that the cytokines produced by the host
in vivo
influence the defense against
C. parvum
infection (
39
). However, our findings demonstrate that
in vitro
, BRV infection inhibits
C. parvum
infection via a mechanism independent of type I and type II IFNs. To explore a potential epithelial-level mechanism underlying this IFN-independent inhibition, we focused on rotavirus enterotoxin with a well-characterized biologically active region.
We identified the NSP4_114–135 peptide of BRV as a potential inhibitory factor against
C. parvum
infection. Because this peptide has been reported to act as a non-competitive and specific inhibitor of the Na
-D-glucose symporter (SGLT1) (
14
), is consistent with a mechanism that could account for the reduced
C. parvum
infection observed in our
in vitro
experiments. NSP4 is the only rotavirus protein known to function as an enterotoxin and induces diarrhea by activating the phospholipase C–inositol trisphosphate pathway upon release from infected cells, resulting in Ca
2+
efflux from the endoplasmic reticulum and enhanced Cl
secretion via Ca
2+
-dependent Cl
channels (
40
). In addition to its enterotoxic activity, the NSP4_114–135 peptide is a fully non-competitive inhibitor of SGLT1 (
14
). Inhibition of SGLT1 has been reported to block
C. parvum
infection by preventing microvillus expansion required for parasite invasion (
41
). Consistent with this mechanism, no significant reduction in
C. parvum
infection was observed at 3 h post-infection, when the parasite is primarily in the adhesion and early invasion stage. These findings are consistent with the possibility that the NSP4 peptide interferes with later stages of host cell invasion rather than initial attachment. In this study, we focused on NSP4 because it is the only rotavirus protein known to directly alter epithelial transport and barrier function. Although other rotaviral proteins may indirectly influence
C. parvum
infection through modulation of host immune responses, direct interference with host cell transporters is most plausibly mediated by NSP4. Future studies will be required to directly test this hypothesis
in vivo
, for example by measuring NSP4 levels and SGLT1 activity in intestinal tissues from mono-infected and co-infected calves, or by experimentally manipulating rotavirus infection prior to
C. parvum
challenge.
In co-infection experiments
in vitro
using human intestinal epithelial cells and
in vivo
in mice, co-infection with human rotavirus WI61 strain and
C. parvum
resulted in a decreased number of rotavirus-infected cells when
C. parvum
infection preceded rotavirus infection, without affecting the severity of diarrhea (
42
). The authors attributed this to the induction of host antiviral immune responses by
C. parvum
-associated dsRNA virus (
Cryptosporidium parvum
virus 1, CSpV1). In Japan, CSpV1 was detected in both
C. parvum
isolates from calves and the experimentally used HNJ-1 strain used in this study (
43
). This observation highlights that
C. parvum
itself can harbor viral elements, supporting the broader concept that virus–parasite interactions may influence parasite biology and pathogenicity, as suggested by our findings for BRV–
C. parvum
co-infection. These findings indicate that co-infection with BRV and
C. parvum
involves complex interactions that are influenced by the sequence of infection and the presence of persistent
C. parvum
infections.
Cryptosporidiosis is a substantial issue in calves, as even a few oocysts (500 oocysts) can establish infection (
44
). In endemic farms, nearly 100% of calves develop cryptosporidiosis; however, clinical symptoms due to
C. parvum
are typically limited to the pre-weaning period. Therefore, mitigating the severity of cryptosporidiosis during this period is crucial for improving cattle productivity. Our findings indicate that BRV–
C. parvum
co-infection is associated with a shortened duration of diarrhea in calves and that BRV NSP4 inhibits
C. parvum
infection
in vitro
, raising the possibility that viral-parasite interactions could be exploited for future control strategies for cryptosporidiosis though a direct
in vivo
role of NSP4 remains to be demonstrated. In farms where BRV is endemic, maternal vaccination against BRV or adequate colostrum administration may contribute to the control of
C. parvum
. Furthermore, approaches that modulate host responses through viral components, such as NSP4 peptides or viral nucleic acids, warrant further investigation as potential measures against
C. parvum
infection. In this study, we acknowledge the limitation of the small number of calves examined, particularly the low number of animals showing natural co-infection. Conducting controlled infection experiments in calves is ethically and logistically challenging, and our analysis was therefore based on naturally occurring infections. In such field-based settings, it is not feasible to manipulate infection timing or repeat sampling, as some animals may not develop co-infection or may be sold before longitudinal data can be obtained. These
in vitro
analyses provided complementary evidence that supports the field observations and strengthens the overall interpretation of rotavirus-induced modulation of
C. parvum
infection. The limited number of calves examined may reduce the statistical robustness of our findings; however, the consistent trends observed in both field and cell-culture experiments support the validity of our conclusions. Recently, it has been reported that the intestinal fungal community of calves influences the infectivity of
C. parvum
45
).
These findings suggest that interactions among multiple microorganisms can regulate pathogenic infections, providing important insights for the development of future infection control strategies. Future studies should further elucidate the mechanisms underlying BRV and
C. parvum
co-infection in cattle.
Statements
Data availability statement
The original contributions presented in the study are included in the article/
Supplementary material
, further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was approved by the Ethical Committee of the Committee on Animal Experiments of the Kyoto Prefectural University of Medicine. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
FM: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing. MI: Investigation, Resources, Writing – review & editing. RS: Investigation, Writing – review & editing. TM: Methodology, Resources, Supervision, Writing – review & editing. KS: Investigation, Resources, Writing – review & editing. TN: Methodology, Writing – review & editing. KK: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was supported by Grants-in-Aid for Scientific Research (B) 24K01921, Scientific Research (C) 22K08584 from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan, by a Livestock Promotional Subsidy from the Japan Racing Association and a grantin-aid from Public Promoting Association Asano Foundation for Studies on Medicine.
Acknowledgments
We thank Dr. Makoto Matsubayashi (Osaka Prefecture University, Japan) for providing the
C. parvum
HNJ-1 strain used in this study. We thank Dr. Kunitoshi Imai (Obihiro University of Agriculture and Veterinary Medicine, Japan) for teaching us the methods for handling rotavirus. We also express our gratitude to Dr. Hisashi Aso (Tohoku University, Japan) for providing BIE cells.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary material for this article can be found online at:
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Summary
Keywords
bovine rotavirus
cattle
co-infection
Cryptosporidium parvum
enteric infection
Citation
Murakoshi F, Itoh M, Soliman RM, Masatani T, Shibano K, Nakaya T and Kato K (2026)
Influence of
Cryptosporidium
and rotavirus co-infection on infectivity in calves
Front. Vet. Sci.
13:1715161. doi:
10.3389/fvets.2026.1715161
Received
29 September 2025
Revised
12 January 2026
Accepted
22 January 2026
Published
17 February 2026
Volume
13 - 2026
Edited by
Vikrant Sudan
, Guru Angad Dev Veterinary and Animal Sciences University, India
Reviewed by
Qingxia Wu
, Tibet Agricultural and Animal Husbandry University, China
Massimiliano Bergallo
, University of Turin, Italy
Roman Fornesa
, University of the Philippines Los Banos, Philippines
Updates
© 2026 Murakoshi, Itoh, Soliman, Masatani, Shibano, Nakaya and Kato.
This is an open-access article distributed under the terms of the
Creative Commons Attribution License (CC BY)
. The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Correspondence: Kentaro Kato,
kentaro.kato.c7@tohoku.ac.jp
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.
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