Retrospective detection of ITGB7 gene mutation in a holstein calf with chronic diarrhea that was suspected of hereditary cholesterol deficiency

Abstract

A homozygous individual for ITGB7 gene mutation, an autosomal recessive congenital disorder in Holstein cattle, was retrospectively identified by genotyping of 195 stored blood from patients less than 12 months of age. Other 24 patients (12.3%) showed heterozygous. The homozygous individual was a 107-day-old female calf born on March 2017, who presented with chronic diarrhea and severe hypocholesterolemia suggesting hereditary cholesterol deficiency (CD), but genotyping analysis showed negative for CD. The patient showed watery diarrhea, dehydration, and extreme emaciation. Necropsy revealed no apparent cause of chronic diarrhea. Histopathological examination revealed mild mucosal inflammation from the jejunum to the colon. Seven years after the patient’s death, the availability of ITGB7 gene mutation testing revealed the patient to be homozygous.

Bovine lymphocyte intestinal retention defect (BLIRD) is a newly identified autosomal recessive disorder in Holstein cattle reported in 2023 [1]. It is caused by a mutation in the ITGB7 gene, which prevents CD4 T lymphocytes from remaining in the intestinal lamina propria [1]. This leads to gastrointestinal immunodeficiency and is thought to result in reduced resistance to parasites [1]. This mutation was identified in a project to identify new genetic defects by comparing SNP data derived from a large-scale genomic evaluation of dead and healthy cattle [1]. Since homozygotes of the ITGB7 gene mutation are not lethal, they have been detected in genetic monitoring tests of slaughtered cattle aged 2 to 10 years in our preliminary study [data not shown]. There have been no reports available for the homozygous mutation of the ITGB7 gene in Holstein cows in Japan due to the lack of a detection system for this disease until recently. Thus, clinical features of the disease and its impact on productivity are unknown. Here, we report the first clinical case of BLIRD in Japan in a Holstein calf with chronic diarrhea and emaciation, with a definitive diagnosis made seven years after necropsy.

The materials of this study were 195 peripheral blood samples collected with ethylenediaminetetraacetic acid-anticoagulated tubes from Holstein patients under 12 months of age stored at −30°C until March 2024, among those provided to Obihiro University of Agriculture and Veterinary Medicine (OUAVM) for the purpose of definitive diagnosis between November 2014 and July 2019. The195 cases were referred to the university hospital of OUAVM for suspected diseases: 44 with cardiac diseases, 7 with respiratory diseases, 54 with digestive diseases, 10 with urogenital diseases, 28 with musculoskeletal diseases, 34 with neurological diseases, 5 with eye diseases, 5 with hematological diseases, and 8 with skin diseases. The age of the 195 cases ranged from 1 to 347 days, with a median age of 40 days. There were 26 males and 169 females. All of these cases were transferred to the university with the consent of the cattle owners. Physical examination, complete blood counts, blood chemical analysis, other specific examination, and necropsy were finally performed under approval Nos. 26-48 (2014), 27-66 (2015), 28-33 (2016), 29-38 (2017), 18-32 (2018), and 19-144 (2019) from the Animal Experiment Committee of OUAVM.

DNA extraction from blood and genotyping were performed at Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, from March to June 2024. Genotyping was performed using Multiplex polymerase chain reaction with sequence-specific primers (PCR-SSP), using fluorescent primers. PCR-SSP is based on SSPCE [3], in which the detection length of elongated DNA amplified using varied sizes of fluorescence primers matched or mis-matched with the 3′-terminus at the target SNP site.

Only one of the 195 (0.5%) analyzed blood samples was shown to have a homozygous mutation in the ITGB7 gene. Other 24 patients among 195 (12.3%) showed heterozygous, which including 4 with ventricle septal defect, 1 with pericarditis, 1 with laryngeal abscess, 1 with coccidiosis, 1 with congenital rectovaginal fistula, 1 with free-martin, 2 with anemia, 1 with thrombocytopenia, 2 with arthritis, 1 with vertebral abscess, 1 with spina bifida, 1 with hydrocephalus, 1 with meningoencephalitis, 1 with congenital cataract, 2 with skin tumors and 3 with neurological symptoms of unknown cause, were found to be heterozygous. The remaining 170 cases among 195 were determined to be wild type.

The homozygous individual was from a 107-day-old Holstein female calf born in March 2017, which presented to a local veterinarian with chronic diarrhea and poor growth (day 1). Instead of treatment with sulfonamide, berberine tannate and infusion of Riger’s solution, no improvement of the diarrhea had found until day 16. Given severe hypocholesterolemia (8 mg/dL) found in the blood test on day19, the local veterinarian suspected hereditary cholesterol deficiency (CD), an autosomal recessive disorder in Holsteins [4, 6, 7]. The patient was presented to Veterinary Teaching Hospital at Obihiro University of Agriculture and Veterinary Medicine (OUAVM) on day 23 for further examination including genotyping analysis. At the time of arrival, the patient had normal appetite and activity, but dehydration, extreme emaciation, and watery diarrhea with a dark green color were observed (Fig. 1). No nematode eggs were found on fecal examination. Although there were no abnormalities in the blood count, the patient still exhibited severe hypocholesterolemia (14 mg/dL), hyponatremia (130 mEq/L), and hypochloremia (92 mEq/L) (Table 1) on Day 23. After notification of the negative result for CD based on the genotyping test [9] on day 27, both the veterinarian and owner requested necropsy to determine the cause of chronic diarrhea.

Fig. 1.

(A) The case showed extreme emaciation. (B) Watery diarrhea with a dark green color were observed.

Table 1. Blood and blood chemical findings (130-day-old).

Test	Results	Reference range	Test	Results	Reference range
RBC (×106 /µL)	10.12	5.1–7.6 [2]	TP (g/dL)	5.7	6.7–7.5 [5]
Hb (g/dL)	10.0	8.5–12.2 [2]	Albumin (g/dL)	2.9	3.0–3.6 [5]
Ht (%)	32.5	22–33 [2]	A/G	1.05	0.83–1.22 [5]
MCV (fL)	32.1	38–50 [2]	AST (U/L)	52	78–132 [5]
MCHC (g/dL)	30.8	36–39 [2]	ALP (U/L)	149	–200 [5]
Platelet (×104 /µL)	80.7	20–65 [2]	GGT (U/L)	16	6.1–17.4 [5]
WBC (/µL)	11,700	5,000–12,000 [2]	LDH (U/L)	726	692–1,445 [5]
Stab (/µL)	117	Rare [2]	CK (U/L)	127	35–280 [5]
Segment (/µL)	2,457	1,800–6,300 [2]	BUN (mg/dL)	5.1	6–27 [5]
Lymphocyte (/µL)	8,775	1,600–5,600 [2]	Total cholesterol (mg/dL)	14	65–220 [5]
Monocyte (/µL)	0	0–800 [2]	Na (mEq/L)	130	132–152 [5]
Eoshinophil (/µL)	351	0–900 [2]	Cl (mEq/L)	92	95–110 [5]

RBC: red blood cells, Hb: hemoglobin, Ht: hematocrit, MCV: mean corpuscular volume, MCHC: mean corpuscular hemoglobin concentration, WBC: white blood cells, TP: total protein, A/G: albumin to globulin ratio, AST: aspartate aminotransferase, ALP: Alkalin phosphatase, GGT: gamma-glutal transferase, LDH: lactate dehydrogenase, CK: creatine kinase, BUN: blood urea nitrogen.

The patient was euthanized for necropsy at the age of 136 days (day 29) by arterial blood exsanguination under deep anesthesia with xylazine (Xylazine Injection 2% “Fujita,” Fujita Pharmaceutical Co., Ltd., Tokyo, Japan) and thiamylal sodium (Isozol, Nichi-Iko, Co., Ltd., Toyama, Japan). Necropsy revealed severe emaciation, reduced intraperitoneal fat tissue, and thymic involution. However, no abnormalities were observed in the external appearance of the alimentary tract. Mild reddish discoloration was observed on the mucosal surface of the jejunum. The cause of chronic diarrhea and emaciation could not be determined at the time of necropsy.

Histopathological examination revealed mild inflammation with crypt abscess formation from the jejunum to the colon, and whipworm parasitism was confirmed in the cecal mucosa (Fig. 2A). Lymphoid follicles in the ileum were small and lymphocytes were scarce in number, especially at the periphery of the follicles (Fig. 2B). However, the size of lymph follicles and the number of lymphocytes differed from the site of the intestine and cases even in the control calf. Immunohistochemical staining using T lymphocyte marker CD3 (mouse monoclonal antibody; Dako, Glostrup, Denmark) revealed that T lymphocytes also distributed in the lamina propria of the intestine in the present case (Fig. 2C). As the anti-CD3 antibody used in this study is a pan-T cell marker, the distribution of CD4-positive cells should ideally be examined. Because decrease in the CD4 detection rate in archived sections by immunohistochemical staining has been reported [12], the reliability on paraffin sections for CD4 has not been guaranteed. Although the T cells observed in the lamina propria of the intestine in this study may be CD8-T cells, future studies using fresh material will be required to address abnormality in the distribution of CD4-helper T cells.

Fig. 2.

Histopathological findings of small intestine. (A) Inflammatory cell infiltration with crypt abscess formation in the lamina propria of jejunum. Hematoxylin and eosin staining. (B) The size of lymphoid follicles in the ileum are small and lymphocytes are scarce in number, especially at the periphery of the follicles. Hematoxylin and eosin staining. (C) CD3 positive T lymphocytes are distributed in the lamina propria of jejunum. Immunohistochemistry.

The first clinical case of CD was reported in 2017 in Japan [11]. Since then, several homozygous and heterozygous CD cases with cholesterol deficiency had been reported by 2020 [4, 8, 10]. At that time, a cholesterol concentration less than 10 mg/dL was considered one of the criteria for suspecting CD [4]. The main clinical signs of the present patient with BLIRD−chronic diarrhea, emaciation, and poor growth−were similar to those of CD [4]. In the present case, we suspected CD at the first stage, but genotyping analysis revealed the patient to be negative for CD, and the cause of chronic diarrhea and emaciation could not be determined at that time. Necropsy also revealed no characteristic findings other than emaciation. Whipworms were confirmed in tissue examination, but their significance in the pathology was unclear.

In BLIRD patients, CD4 T lymphocytes cannot remain in the lamina propria mucosa, which is thought to weaken the defense against intestinal infections and increase the susceptibility to infectious enteritis, including parasitic infection [1]. Although chronic diarrhea and emaciation may result in a decrease in productivity, they do not necessarily lead to immediate death. There are several diseases that cause chronic diarrhea in young cattle and lead to poor growth. Calves with unknown causes of chronic diarrhea, emaciation, and poor growth are often diagnosed with “weak calf syndrome,” and in such cases, their growth is delayed even when they become heifers. BLIRD is thought to be one of the true causes of weak calf syndrome.

In the present case, the cause was unknown at the time of necropsy in 2017, but seven years later, genetic analysis led to a definitive diagnosis of BLIRD. This is the first confirmed case of a homozygous BLIRD calf in Japan. The clinical feature of the patient was remarkably close to that of CD. The disease should be suspected in growing calves and heifers with poor growth, weakness, and chronic diarrhea of unknown etiology [1]. It is also important to note that since there are many causes of diarrhea, it has not been conclusively proven that the clinical symptoms in this case are only related to the ITGB7 gene mutation.

In this study, 12.3% of patients with various clinical symptoms other than chronic diarrhea showed heterozygous for the ITGB7 gene mutation, suggesting that this gene mutation is widely distributed in Holstein dairy cows in Japan. To characterize precise clinical and pathological features of BLIRD, and to determine the economic losses resulting from the disease, further studies are needed to examine the productivity and health records of homozygous and heterozygous carriers of BLIRD.

POTENTIAL CONFLICTS OF INTEREST

The authors have nothing to desclose.