key: cord-1050454-mkxb0bsf
authors: Dahmani, Hichem; Ouchene, Nassim; Dahmani, Ali; Ouchene-Khelifi, Nadjet Amina
title: First report on Cryptosporidium parvum, Escherichia coli K99, rotavirus and coronavirus in neonatal lambs from north-center region, Algeria
date: 2020-10-22
journal: Comp Immunol Microbiol Infect Dis
DOI: 10.1016/j.cimid.2020.101567
sha: d026e46fc0ed94fe0d71f6289f386e826afa6cf9
doc_id: 1050454
cord_uid: mkxb0bsf

The etiology of neonatal diarrhea is multifactorial and remains one of the greatest health problems in sheep livestock farming. Faecal samples from 559 neonatal lambs aged less than 30 days from 30 sheepfolds located in the north-center region of Algeria were screened with pathogen-specific antigen ELISA for Cryptosporidium parvum, Escherichia coli K99, rotavirus, and coronavirus. Of the 559 lambs, 312 (58.81%), 155 (27.72%), 72 (12.88%) and 20 (3.57%) were positives for C. parvum, E. coli K99, rotavirus and coronavirus antigens, respectively. The prevalence of C. parvum was the highest (p < 0.0001). C. parvum, E. coli K99, rotavirus and coronavirus were observed in 23 (76.66%), 17 (56.66%), 9 (30%) and 3 (10%) sheepfolds, respectively. Compared to age, the prevalence of C. parvum was highest during the second and third week of age (p < 0.001). In contrast, other pathogens were found to be more frequent in lambs aged ≤7 days (p < 0.001). The number of lambs with diarrhea was 280 (50.09%) of which 280 (100%), 127 (45.35%), 52 (18.57%) and 10 (3.57%) were found to be infected with C. parvum, E. coli K99, rotavirus and coronavirus, respectively (p < 0.0001). In various combinations, mixed infections were detected only with C. parvum. This is the first report of C. parvum, E. coli K99, rotavirus, and coronavirus in ≤30-days old neonatal lambs in Algeria. Special attention should be given to the first colostrum feeding, hygiene of the farm, prevention and control measures for a better prevention of neonatal diarrhea in lambs.

Neonatal diarrhoea remains one of the greatest health problems in ruminant livestock production, resulting in significant economic losses due to morbidity and mortality, treatment costs and reduced growth rates of affected newborns (Garaicoechea et al., 2006; Reidy et al., 2006) . Its etiology is multifactorial, resulting from an interaction between the animal and its environment, nutrition and the pathogens involved (Scott et al., 2004) .

The etiology of this diarrhoeal syndrome can be infectious (viruses, bacteria and protozoa) or due to non-infectious factors such as herd management, nutritional and immunological status of the host (Garaicoechea et al., 2006; Maes et al., 2003; Stipp et al., 2009) . Infectious diarrhoea in lambs is most often due to different types of enterotoxigenic Escherichia coli, Cryptosporidium parvum, Rotavirus, and Coronavirus or a combination of these pathogens.

They can cause clinical disease and/or may lead to suboptimal growth rate of affected animals (Chatzopoulos et al., 2013; Hubalek et al., 2014; Minetti et al., 2014; Scholes et al., 2007 , Yang et al., 2009 .

However, in a neonatal diarrhoea syndrome, it is difficult to situate the exact etiological role of each of these pathogens, due to their action which can be isolated or associated, producing practically similar clinical signs. Moreover, epidemiological studies have shown that the diversity in the degree of association between these agents is often fatal when the infections are mixed (Chatzopoulos et al., 2013; Hubalek et al., 2014; Martella et al., 2015) .

In Algeria, the overall sheep population was estimated at 28,393,602 (MADR, 2017).

Although, neonatal diarrhoea is one of the major preoccupations of livestock farmers in Algeria. However, to date no studies have been carried out in Algeria to investigate the main agents of neonatal diarrhoea in sheep (Cryptosporidium parvum, Escherichia coli K99, rotavirus and coronavirus). The majority of publications have only concerned cattle (Khelef et J o u r n a l P r e -p r o o f Ouchene et al, 2012 Ouchene et al, , 2016 and the very few studies that have been carried out on sheep are concerned only Cryptosporidium spp (Baroudi et al, 2018; Sahraoui et al, 2019) .

The present study was therefore conducted for evaluate the comparative role of Cryptosporidium spp and other enteric pathogens known as major agents of neonatal diarrhea in lambs on 30 farms located in the north-central region of Algeria.

The study was carried out between January 2014 and December 2016 and concerned 559 lambs aged less than 30 days. Lambs were classified into four age groups: ≤ 7 days, 8-14 days, 15-21 days, 22-30 days.

The study was carried out in 30 sheepfolds with extensive farming methods, where sheep remain on pasture for the most part of the day. The study was carried out in north-center region of Algeria: Laghouate (33°47′59″ N 2°51′54″ E), Djelfa (34°40′22″ N 3°15′46″ E), Medea (36°15′51″ N 2°45′14″ E) and Bouira (36°22′29″N 3°54′07″ E) ( Figure 1 ). Fecal samples were taken using a rectal swab from each lamb in a sterile plastic vial. The samples were then transported to the laboratory at 4°C. Farm of origin, the date of sampling and the age of each animal were recorded. The aspect of the faeces was registered: diarrhoeal/non-diarrhoeal.

Faecal samples were screened for antigens to C. parvum and the other enteric pathogens using a commercial faecal antigen ELISA test (Bio-X Easy-Digest, Bio K 151; Bio-X Diagnostics, Belgium). This kit simultaneously detects specific antigens from C. parvum, E. coli K99, rotavirus, and coronavirus and the tests were performed according to the kit manufactures' instructions.

Detection rate of prevalence in diarrheic lambs than in no-diarrheic and in the different age groups were compared by chi-square test (χ2) test. For multi-variable comparisons, one-way ANOVA was conducted, followed by Tukey-Kramer testing using the program R software version 3.0.1(R Core Team2013). Differences were considered significant at p˂0.05.

Of the 559 lambs, 312 (58.81%), 155 (27.72%), 72 (12.88%) and 20 (3.57%) tested by faecal antigen ELISA were positive for C. parvum, E. coli K99, rotavirus and coronavirus antigens, respectively. The prevalence of C. parvum was the highest (p<0.0001) ( Table 1) .

C. parvum, E. coli, rotavirus and coronavirus were observed in 23 (76.66%), 17 (56.66%), 9 (30%) and 3 (10%) sheepfolds, respectively. C. parvum was the most widespread (p<0.0001) .

Compared to age, the prevalence of C. parvum was highest during the second and third week of age (p<0.001). In contrast, other pathogens were found to be more frequent in lambs aged ≤7 days (p<0.001) ( Table 2 ). Rotavirus and coronavirus were not revealed in lambs aged ≥ 15 days (Table 2) .

Lambs aged ≤ 7 days were found to be more infested with E. coli K99 and rotavirus compared to C. parvum and coronavirus (p<0.001) ( Table 2 ). In the other age categories (≥8jours), lambs were more infested with C. parvum than other enteropathogens (p<0.001) ( Table 2 ).

The number of lambs with diarrhea was 280 (50.09%) of which 280 (100%), 127 (45.35%), 52 (18.57%) and 10 (3.57%) were found to be infected with C. parvum, E. coli K99, rotavirus J o u r n a l P r e -p r o o f and coronavirus, respectively. All diarrheal lambs were found to be infected with C. parvum, which showed a significantly higher prevalence than other pathogens (p<0.0001).

The prevalence of infection with C. parvum, E. coli and rotavirus was higher in lambs with diarrhea compared to lambs without diarrhea (p<0.001). However, no significant difference was observed for coronavirus (Table 3) .

C. parvum, E. coli K99, rotavirus and coronavirus were identified alone in 291, 146, 65 and 15 samples, respectively ( Figure 2 ). C. parvum was the most frequent (p<0.0001).

The association between these pathogens was observed only with C. parvum, where associations of C. parvum with E. coli K99, rotavirus and coronavirus were reported in 9, 7 and 5 samples, respectively ( Figure 2 ).

The clinical diagnosis of infectious enteritis is difficult to establish because of the frequency of clinical signs, the implication of multiple agents, and the interactions between factors that predispose the host to infection (Athanassious et al., 1994) . C. parvum, E. coli K99, rotavirus and coronavirus are recognized as the most important enteropathogens in diarrhea in young ruminants (Gulliksen et al., 2009) . To our knowledge, this is the first report of neonatal lambs in Algeria in which C. parvum along with E. coli K99, rotavirus, and coronavirus infections are reported using the ELISA test for fecal antigen detection. The results showed the existence of all this pathogens with a predominance of C. parvum.

Prevalence of lambs' infections with C. parvum, E. coli K99, rotavirus and coronavirus revealed in this study were 58.81%, 27.72%, 12.88% and 3.57%, respectively. C. parvum was the most prevalent. This is in agreement with other findings (Ahamed et al., 2015; Gencay, 2012; Munoz et al., 1996; Paze et al., 2014) . C. parvum plays an important role in neonatal diarrhea in lambs (Andres et al., 2007) . In our study, a strong association between diarrhea and the presence of C. parvum oocysts in lamb faeces have been recorded which is in concordance with several other studies (Ahamed et al., 2015; Causape et al., 2002; Mueller-Doblies et al., 2008; Munoz et al., 1996; Paoletti et al., 2009; Ulutaş and Voyvoda, 2004) . The same finding was reported also in cattle (Brandão et al., 2007; Izzo et al., 2011; Stipp et al., 2009 ).

C. parvum was observed in 23 sheepfolds (67.66%) which is comparable with Causapé et al.

(2002), Munoz et al. (1996) and higher than of Nouri et al. (1993) in Iran, Rossanigo et al. (1987) in Italy and Causape et al. (2002) and Pablo Diaz et al. (2015) in Spain.

In this study, the higher prevalence of cryptosporidial infection was observed in lambs aged between 8-21 days. The same was reported by Sevinç et al. (2005) . The presence of Cryptosporidium infection in clinically asymptomatic lambs indicated that particular age group of animals might be reservoir for the parasite (Pritchard et al., 2008) and the prevalence rate decreases with increase in age and, this may be due to age related immunity (Zu et al.,1992) .

Neonatal colibacillosis is highly contagious in lambs (Pritchard et al., 2007) . Our results show that E. coli K99 was reported in 27.72% of lambs which is consistent with 26% reported in Spain (Munoz et al., 1996) and higher than 19.2%, 9.42% reported by Fernando et al. (2016) in Brazil and Malik et al. (1983) in Morocco, respectively.

The most severe diarrhea was recorded in calves less than three days old with E. coli K99 (King et al., 1991) . In our study, the highest prevalence of E. coli K99 was observed in lambs less than 7 days old which is in agreement with Erhan et al. (2010) .

E. coli K99 was identified in 81.93% of the diarrhoeal lambs in our study which is in agreement with Aklilu et al. (2013) in Etiopia (84%). E. coli K99 was isolated from 4% of lambs in the Netherlands (Heuvelink et al., 1995), 0.2% in Italy (Bettelheim and Beutin, 2003) , 31% in USA (Keenan et al., 1986) from sheep flock and between 12.5-26.6% (Bhat et al., 2008; Wani et al., 2003) in India. These reports are much lower than our finding.

The high prevalence of E. coli K99 observed in our study can be attributed to delayed or insufficient in first colostrum intake, unclean shepherd and lack of implementation of appropriate prevention and control measures. Olsson et al. (1993) reported that each hour of delay in colostrum ingestion during the first 12 hours of age resulted in a 10% increased risk of a lamb becoming sick. The same observation was suggested by Aklilu et al. (2013) .

Lambs are susceptible to different rotavirus serotypes during their first week of life (king et al., 1991) . Rotavirus-associated enteritis in lambs has been the subject of numerous studies; the frequency of infection ranges from 2.1% in Spain (Munoz et al., 1996) and 5.3% in Turkey (Erhan et al., 2010 ) to 25% in India (wani et al., 2004 . In our study, the prevalence encountered (12.88%) falls between these prevalences.

In the present study, rotavirus was more observed in lambs less than 7 days old (35.48%). The quantitative and qualitative insufficiency of colostrum can be incriminated, especially since double births are frequent in ewes (Theil et al., 1995) .

Several studies have shown high morbidity (75-100%) in outbreaks of neonatal diarrhea in lambs (Papp et al., 2014) . In the present study, rotavirus was more prevalent in diarrhoea than non-diarrhoeal lambs, which is consistent with Kaminjolo et al. (1994) .

Rotavirus was isolated from 30% of the farms in our study, which is lower compared to 46% found in Scotland (Wani et al., 2004) .

Coronavirus has been reported to play a role in the etiology of diarrhoea in association with rotavirus (Chapman et al., 2001; Ozmen et al., 2006) . In the present study, coronavirus was detected in 3.57% of lambs, which was lower than reported by other authors (Burimuah et al., 2020; Erhan et al., 2010) .

In addition, a survey of small ruminants in Spain failed to detect coronaviruses in diarrhoeal neonates (Munoz et al., 1996) .

Many enteric pathogens can exist simultaneously with cryptosporidium infection in the same animal (Munoz et al., 1996) . Although infections with C. parvum alone were recorded, mixed infections with the other enteric pathogens tested were more frequent. As demonstrated also, single infections with E. coli K99, rotavirus and coronavirus may be associated with diarrhoea (Cho and Yoon, 2014) . For mixed infections, C. parvum has been variably detected with E. coli K99, rotavirus and coronavirus. These mixed infections are common and are generally thought to increase the severity of the diarrhea when they occur (Cho and Yoon, 2014) .

In our study, mixed infections were observed between C. parvum and E. coli K99, rotavirus or coronavirus and no other form of association was found. This suggests that C. parvum is a primary agent in neonatal diarrhoea in lambs and that other agent is synergistic. The same observation has been reported by other authors (De la Fuente et al., 1999; Tartera et al., 2000) .

The present study has documented C. parvum, as well as E. coli K99, rotavirus, and coronavirus in neonatal lambs from Algeria. These enteropathogens were observed in lambs with or without diarrhea. C. parvum was the most common, especially in lambs with diarrhea. 

Prevalence and analysis of associated risk factors for Cryptosporidium infection in lambs in Jammu district

Identification and Biotyping of

Escherichia coli from Diarrheic Lambs in and Around Debrebirhan Town, Ethiopia

Evaluation of some etiological factors predisposing to diarrhoea in lambs in

Detection of bovine coronavirus and type A rotavirus in neonatal calf diarrhea and winter dysentery of cattle in Quebec: evaluation of three diagnostic methods

Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria

Rotavirus infection in domestic animals

Cryptosporidium and Concurrent Infectious with Other Major Enteropathogenes In 1 to 30 day old diarrheic dairy calves in central Spain

Enteric Pathogens in the Aetiology of Diarrhoea in Neonatal Lambs

Lambs are an important source of atypical enteropathogenic Escherichia coli in southern Brazil

Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period

Sheep as an important source of E. coli O157/O157: H7 in Turkey

Enteropathogens and risk factors for diarrhea in Norwegian dairy calves

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe

Arboviruses pathogenic for domestic and wild animals

Prevalence and analysis of associated risk factors Cryptosporidium infection in lambs in Jammu district

Prevalence of major enteric pathogens in Australian dairy calves with diarrhoea

Rotavirus infection in calves, piglets, lambs and goat kids in Trinidad

Morphologic evaluation of the effects of Shiga toxin and E coli Shiga-like toxin on the rabbit intestine

Épidemiologie de la cryptosporidiose chez les bovins en Algérie

Neonatal diseases in Diseases of sheep

N°837

Evaluation of a human group A Rotavirus assay for on-site detection of bovine rotavirus

Les entérites à E. coli K99 chez le veau et l'agneau nouveaux nés: Etude épidémiologique

Enteric viral infections in lambs or kids

Occurrence and diversity of Giardia duodenalis assemblages in livestock in the UK

Distribution of Cryptosporidium species in sheep in the UK

Role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in Spain

Effect of nomada shepherds and their sheep on the incidence of cryptosporidiosis in an adjacent town

Calf diseases and mortality in Swedish dairy herds

Prevalence of Cryptosporidium sp in dairy calves in north-eastern Algeria

Observations and immunohistochemical detection of coronavirus, Cryptosporidium parvum and Giardia intestinalis in neonatal diarrhoea in lambs and kids

Cryptosporidium species and subtype analysis in diarrhoeic pre-weaned lambs and goat kids from north-western Spain

Prevalence of Cryptosporidium infection in sheep flocks with a history of lambs' diarrhoea

Immunoenzymatic analysis and genetic detection of Cryptosporidium parvum in lambs from Italy

Rotavirus strains in neglected animal species including lambs, goats and camelids

High Occurrence of Cryptosporidium ubiquitum and Giardia duodenalis genotype E in sheep from Brazil

Cryptosporidium species in lambs submitted for diagnostic postmortem examination in England and Wales

Cryptosporidium parvum infection in orphan lambs on a farm open to the public

Molecular characterization and analysis of bovine rotavirus strains circulating in Ireland

FloroniA,Rivero VB. Diagnosi di criptosporidiosi in alcunialle. vamentidell' Italia Centrale

Molecular characterization of zoonotic Cryptosporidium spp. and Giardia duodenalis pathogens in Algerian sheep

Clostridium perfringens type D enterotoxaemia in neonatal lambs

Calf Diarrhoea

The Prevalence of Cryptosporidium parvum in Lambs around Konya

Frequency of BCoV detection by a semi-nested PCR assays in faeces of calves from Brazilian cattle herds

Quand Suspecter la cryptosporidiose ? La Semaine Vétérinaire

Group B rotavirus associated with an outbreak of neonatal lamb diarrhea

Cryptosporidiosis in Diarrhoeic Lambs on a Sheep Farm

Epidemiology of diarrhoea caused by rotavirus and Escherichia coli in lambs in Kashmir valley

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India: The Society for Applied Microbiology

Longitudinal prevalence, faecal shedding and molecular characterisation of Campylobacter spp. and Salmonella enterica in sheep

Prevalence and molecular characterisation of Cryptosporidium and Giardia species in pre-weaned sheep in Australia