Pathogens in faeces of calves aged from

1-21 days in Dutch dairy herds;

Prevalence and risk factors.

Menno Holzhauer, Chris Bartels, Sylvia Blom (veterinary student),

Martin Oldenhof (student agricultural science), Ton de Gee

Dutch Animal Health Service, P.O. Box 9, 7400 AA Deventer, The Netherlands

By order of: Schering-Plough, SA/NV

P.O. Box 1364

3600 BJ Maarsen

The Netherlands

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Summary

Within the period of January-April 2007 424 calves from 1-21 days in 108 Dutch dairy

herds, were sampled to investigate the prevalence of diarrhoea and the pathogens in de faeces as

regards number and factors related to animal and herd). The specific pathogens investigated,

were: Clostridium perfringens (C. perfringens), Corona virus, Cryptosporidium parvum (C.

parvum), Eschericia coli K99 (E. coli) and Rota virus. Apart from that, information was collected

about each individual calf (age, sex, temperature, extent of tenting, heartbeats per minute) and

the herd management (housing, (way of and litres) supply of colostrums and milk, treatment of

diarrhoea). This information was analysed separately in relation to faeces consistency and the

presence or absence of a special intestinal pathogen.

Fifty seven percent of calves had faeces of normal colour (brownish) and consistency

(firm), 24% of the calves had yellow colourized faeces, with custard consistency and 19% of the

calves had watery-like faeces. Infection with C. parvum was the most important pathogen for

faeces at variance, both for yellow colourized faeces, with custard consistency and faeces of watery

consistency. The prevalence for E. coli was 2.6% (95% confidence interval (CI) 1.3-4.6%), for

Corona virus 3.1% (95% CI: 1.6-5.2%), for Rota virus 17.7% (95% CI: 14.2-21.7%), for C.

parvum 27.8% (95% CI: 23.6-32.4%) and for Cl. perfingens 54.0% (95% CI: 49.1-58.8%). E. coli

was present in 10 herds (=9%), Corona virus in 13 herds (=12.%), Rota virus 50 herds (=46%),

C. parvum in 62 herds (=57%) and Cl. perfingens in 92 herds (=85%). In case of presence of E.

coli and Corona virus the agents were found in only 1 or 2 calves of a herd at maximum, while Cl.

perfingens and C. parvum were more frequent diagnosed in more calves within the same herd.

Based on a logistic regression analysis the significant risk factors for E. coli were: the presence of

under temperature (temperature < 38.5 functioned as a preventive factor), presence of Corona

virus in the herd and age of the calf (mainly 1st week after birth). Risk factors for Corona virus

were: moderate hygiene in the herd and the presence of E. coli and C. parum in a herd. Risk

factors for C. parvum were: moderate hygiene in the herd, presence of Rota virus in a herd and

age (mainly 2nd week). Risk factors for Rota virus were: number of calves present, number of

diarrhoeic calves and age after birth also (mainly 2nd week). For Cl. Perfringens only a relation with

age was estimated (mainly first week).

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Introduction

A good rearing of young stock ought to result in well developed heifers, which are optimally

prepared for such a good production that the genetic potency will come to expression completely.

Well developed heifers will produce better than less developed herd mates (Subnel et al., 1994).

Most important causes of young calves’ health problems (0 – 1 year of age) are intestinal and

respiratory problems (Waltner-Toews et al., 1986a; Kaneene and Hurd, 1990). Especially diarrhoea

during the first month of life in dairy calves is responsible for serious direct and indirect economical

damage and consequently will result in an increased use of medicines in dairy herds (Tzipori, 1981;

Olsson et al., 1999). The indirect economical damage is caused by delayed growth of the calves,

which might also result in calving of the heifers at an older age (de Graaf at al., 1999). Besides

that, a period of diarrhoea makes the calves more sensitive for (infective) respiratory problems

during the 2nd and 3rd month of age. The highest risk of death of dairy calves is during the first 3

weeks after birth (Waltner-Toews et al., 1986a; Curtis et al., 1988; Perez et al., 1990; Wells et al.,

1996). Waltner-Toews et al. (1986a) found that the peak of diarrhoea treatment was in the 2nd

week. A part of the dead calves is presented at the Dutch Animal Health Service (DAHS)

Laboratory and other institutes for post-mortal investigation. These investigations will result in an

isolation of all the (possible) involved agencies and this information can be used for improving the

herd therapy and prevention. However the calves offered for this investigation have been treated

frequently and/or suffering for a longer period. Consequently the primary (causing) agents are not

isolated anymore.

Diarrhoea in dairy calves is considered to have a multifactorial aetiology, whereby viruses,

bacteria as well as protozoa may play a role (Lorenz, 2006). The resulting illness in relation to

these agencies is in fact the result of both animal (e.g. race and age) and environmental (e.g.

housing, neighbourhood and colostrums (no. of litres and quality) factors.

Escherichia coli K99 (E.coli), rota virus, corona virus and Cryptosporidium parvum (C.

parvum) are responsible for almost 75-95% of the cases of the intestinal infections in young calves

(< 1 month) (Acres, 1985; Tzipori, 1985). In these calves a clear relation was seen between these

presences of these intestinal agencies and the appearance of diarrhoea (Garcia et al., 2000). An

increasing interest can be seen for to the association of toxin producing Clostridium perfringens (Cl.

perfringens) and the resulting enterotoxaemia in both dairy cows and calves (Muskens et al., 2007;

Lebrun et al., 2007). Lebrun et al. (2007) found an association between a typical variant of Cl.

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perfringens producing toxins and the bovine enteroxaemic syndrome. Moreover the selective

advantaging circumstances for this agent have to be cleared up.

A study based on diarrhoeic calves by De Graaf et al. (1999) estimated in Belgium

prevalence for E.coli, rotavirus, coronavirus and C. parvum of respectively 4%, 20%, 8% and 31%.

An investigation in Belgium from the early 20th estimated a C. parvum prevalence of 22% in calves

from 0-4 weeks (Geurden et al., 2005). Comparable prevalence was estimated in Sweden and

Germany (Björkman et al., 2003; Luginbühl et al., 2005).

Apart from infectious causes also non-infectious causes (e.g. bad prepared milk) may be

responsible for diarrhoea in (dairy) calves. Based on clinical investigation no clear differentiation

can be made between the possible causes of diarrhoea. This can only be made with the support of

laboratorial diagnostic techniques (Nussbaum et al., 1999). Also allergic reactions have been

proven to play a role in the aetiology of diarrhoea in young calves (Fledderus et al., 1992).

The aim of this study was to acquire insight into the prevalence of diarrhoea, of 5 intestinal

pathogens and related animal and/or herd-factors in Dutch dairy herds. The results of this study

will supply the farmers with tools for more specialized measures and possibilities for the

accompanying vet to have their (preventive and curative) advice better founded. Based on earlier

results of investigations in DAHS laboratory (e.g. post mortal findings) and Belgium (De Graaf et

al., ’99) whereby pathogens mentioned below were isolated from diarrhoeic calves, this study was

focused on 5 main pathogens:

1. E. coli K99 (further mentioned as E. coli)

2. Rota virus

3. Bovine Corona virus

4. Cryptosporidium parvum (C.parvum)

5. Clostridium perfringrens (Cl. perfringens)

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Material and methods

a. Selection of and performance in the dairy herds

Based on an estimated prevalence of the pathogens of 50%, a minimal estimation of the

size of sampling was calculated for a reliable result. At an expected prevalence of 50%, a 5% range

of error and a confidence interval of 95%, a sample size was calculated of 385 calves. In this

investigation the sample size was for convenience round up to 400 faecal samples.

With a mean herd size of 72 dairy cows in Dutch dairy herds, where every month between

5-6 calves are born, 80 herds were needed to collect 400 faecal samples. To take sufficient herds

and calves into the investigation 309 dairy herds were approached telephonically in The

Netherlands for participation in the investigation for intestinal pathogens in calves between 1-21

days of age. The selection of these herds was at random out of the total stock of more than 20.000

dairy herds in The Netherlands.

In this investigation not only information about prevalence at animal level, it was important

to gather information about the extent of distribution of infection within a dairy herd but also

(within-herd prevalence). Therefore herds with only 1 calf present between ages of 1-21 days were

excluded from the investigation.

The visits to the herds were executed while a fixed protocol was applied (schedule of work

from young to older calves and absence and presence of clinical problems, temperature measuring,

hygiene, disinfection etc.). Minimumally 2 and maximally 6 calves at each herd were included in

the investigation and a rectal faecal sample was taken, whereby for each calf a new plastic glove

was used. In case of the presence of more calves per group of age (1= 0-7 days; 2= 8-14 days;

3= 15-21 days), from each group 2 calves were selected, investigated and sampled randomly. The

faecal samples were collected in a small plastic pot and criticized by the investigator visually on

consistency. After the sampling the unique herd and calf number and date of sampling were written

on the plastic pot and a list and the sample was cooled transported to the lab (DAHS, Deventer,

The Netherlands). Before the sampling, from each calf, information about breed, date of birth,

presence of diarrhoea and treatments in relation to diarrhoea or other illness, was recorded. After

sampling of the calves and at the end of the herd visit a survey was conducted about among

colostrums and feeding management, nursing and housing (individual, in groups or at the cow) of

the calves in the herd (see supplement etc.). Before the start of the real investigation 2 herds were

visited by the two investigators at the same time, to have the performance of the investigation

tuned.

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Diarrhoea was defined as a clearly increased frequency of defecation with relatively higher

water substance and relative decrease of material substance (Radostits et al., 2006). For the

evaluation of the faecal consistency the following criteria were defined: score of faeces = 0, no

diarrhoea, perineum and tail of the calf clean and dry; score = 1, faeces has a custard consistency,

yellow colourized and weak with a higher fraction of water, perineum and/or tail of the calf were

smeared with faeces; score = 2, watery faeces, perineum and/or tail of the calf are smeared with

wet faeces.

b. Laboratorial investigations

Faecal samples were investigated in the DAHS laboratory for the presence/absence of E.

coli (K99/F5), Rota virus, Bovine Corona virus and C. parvum with the use of a tetrakit test

(Tetraquick, Bio-X Diagnostics, Belgium). Investigation for the presence of Cl. perfringens was

based on an anaerobic culture technique on an anaerobic blood agar plate. Cl. perfringens was only

scored as absent or present and not further typed for toxin producing.

c. Statistical analysis

The presence of intestinal pathogens in calves from 1-21 days on dairy herds at a certain

moment (calf prevalence) was estimated as the number of calves whereby the concerned pathogen

was found by faecal examination divided by the total number of sampled calves. The herd

prevalence was estimated as the total number of herds with at least one faecal sample in which the

pathogen was found divided by the total number of herds.

Both at individual animal level and at herd level Spearman-rank correlation coefficients

were estimated for any combination of 2 intestinal pathogens. The correlation coefficients

demonstrate to which extent intestinal pathogens are found simultaneously. The estimated

correlation coefficient range from -1 via ‘0’ (not any correlation) until ‘1’ (full present at the same

time).

To investigate factors for a not-normal faecal consistency a multi-nominal regression

analysis was performed. The outcome variable ‘faecal consistency’ had 3 possible variables (normal

consistency, custard consistency and watery faeces). Based on ‘faecal consistency’ as outcome

variable an analysis was performed to predict which of the investigated pathogens were responsible

for the not normal faecal consistency. For this analysis the data have been corrected for animal

symptoms (age, sex), housing symptoms (iglo, solitary boxes, or clustered housing), milk feeding

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(factory milk, cow milk, bulk milk, solution of electrolytes), animal symptoms (fever,

(temp>39.5

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oC), under temperature (Temp<38.0oC, tenting, different heart frequency) and

application of medication for diarrhoea.

Apart from that per intestinal pathogen a logistic regression analysis has been performed to

identify possible risk factors. Hereby possible ‘confounding variable’ (age, faecal consistency) were

integrated in the model.

For all regression analyses a correction has been carried out for clustering at herd level by

implementing herd as a ‘random effect’ in the model. The extent in which infection is present

within a dairy herd in relation to the presence of an infection between herds was quantified as the

‘intra-class correlation coefficient’ (ICC). The ICC is defined as the correlation between two

observations within a dairy herd. It gives insight to what extent of different factors that play a role

within a herd (housing, colostrums and milk supply, transmission of infection within a herd,

unknown factors) are the cause that a number of calves are infected simultaneously. The ICC value

varies between ‘0’ (no relation between infection in calves on the same dairy herd) and ‘1’ (a

complete relation between calves at the same herd; all calves in that herd have the same

infection). All analyses were performed with STATA/SE 8.2 (Statacorp, 2004). A P-value of ≤0.05

was considered to be a significant association, while a P-value between 0.05-0.10 was considered

to be a statistical indication

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Results

Of the in totally 309 approached dairy herds, 119 herds were visited actually and hereby

464 calves were involved in the investigation.

Reasons for the herds not (being able) to participate in the investigation were:

- No or not sufficient suitable calves at the moment of approach (75%);

- No interest, time for investigation(s) or no problems (20%);

- No telephonic contact, despite several attempts (5%)

After control of the suitability/completeness of the data etc., finally the results of 108 herds and

424 calves were used for further analysis of the questions of investigation. Reasons for loss of data

were among others missing information of the calves, the impossibility to collect a faecal sample

(16x) and/or the (afterwards) not fulfilling the criteria of section of the calves (too young, old etc.)

(24x). Of the 108 investigated herds, 1 herd had a biological signature; all the other herds had a

more traditional design.

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Prevalence of abnormal faeces.

Abnormal faeces were recorded in 2 different options: yellow colourized and a custard-

consistency like (24%) and watery like (19%). Abnormal faeces were most times seen during the

2nd week of ages (8-14 days) (table 1).

Table 1. Presence of normal and abnormal faeces for each group of age in dairy herds (n=424).

Consistency of

faeces

Age

Normal

(N= 242)

Custard consistency

(N=101)

Watery

(N=81)

% of calves for

each group of

age

1-7 days 61.8% 20.8% 17.3% 40.8%

8-14 days 48.4% 24.8% 26.8% 36.1%

15-22 days 62.2% 27.6% 10.2% 23.1%

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Prevalence of intestinal pathogens

Intestinal pathogens were found in different prevalence, both at level of individual calf and

at herd level (Table 2).

The prevalence of the intestinal pathogens in calves varied from a small number for E. coli (2.6%)

and Corona virus (3.1%) until a large number for Cl. perfringens (54.0%). Also at herd level E. coli

(9.3%) and Corona virus (12.0%) were seen in a small number and Cl. perfringens much more

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(85.2%). When E. coli and Corona virus were found in a dairy herd, it was always present in 1 or

max. 2 calves. This was different for the other 3 pathogens. Especially Cl. perfingens and C.

parvum were found frequently in different calves in the same herd.

Table 2. Prevalence (95% confidence interval) of intestinal pathogens in calves (1-21 days)

(n=424) in dairy herds (n=108) and the intra class correlation coefficient.

E.coli Corona Cl. perfringens C. parvum Rota

Calves 2.6%

(1.3-4.6%)

3.1%

(1.6-5.2%)

54.0%

(49.1-58.8%)

27.8%

(23.6-32.4%)

17.7%

(14.2-21.7%)

Herds 9%

(5-16%)

12%

(7-20%)

85%

(77-91%)

57%

(48-67%)

46%

(37-56%)

1 8.3% 12.0% 25.9% 28.7% 30.6%

2 1.0% 24.1% 13.9% 10.2%

3 15.7% 8.3% 3.7%

4 11.1% 4.6% 1.9%

5 3.7% 1.9%

Number of

calves per

herd

6 4.6%

Intra-class correlation

coefficient

27%

not

significant

Not

applicable

24%

(12-40%)

33%

(19-51%)

19%

(7-43%)

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Intra-class correlation coefficient

The way the presence of an infection in calves is influenced by the herd (and consequential the

factors that are determined at herd level, as housing, colostrums and milk supply etc.) is quantified

based on the ICC (table 11). Because E.coli hardly, and Corona virus not at all, was present at

more than 1 calf, the ICC-values are not relevant for these pathogens. For the other 3 intestinal

pathogens it was demonstrated in table 11 that the infections are to a reasonable extent herd

related, with ICC values that vary from 19% for Rota virus until 33% for C. parvum.

At 28% of the calves no pathogens were found at all, while at 25% 2 or more pathogens

were found (table 3). When Cl. perfringens was left aside, at almost 60% of the calves no

pathogens were found and at only 10% 2 or more pathogens.

Table 3. Presence of none, 1 or more intestinal pathogens at calves in dairy herds (n=424)

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No. of intestinal

pathogens

Inclusive Cl.

perfringens

Exclusive Cl.

perfringens

None 28.1% 59.7%

1 46.2% 30.3%

2 18.9% 9.2%

3 6.4% 0.5%

4 0.2% 0.3%

5 0.2% 0.0%

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The combinations where different intestinal pathogens were found at the same time are

presented in table 4. At the level of individual calves, E. coli was significantly more present in

relation with Corona virus. A comparable relation was present for Corona and Rota virus in case of

a C. parvum infection.

Also at herd level, a statistical relation was found between the presence of Corona or Rota

virus combined with a C. parvum infection. Besides E. coli and Corona virus seemed to be present

simultaneously quite frequently. Finally Cl. perfringens was significantly more found in calves with

a Rota virus and C. parvum infection.

Table 4. Two by two relations of the presence of intestinal pathogens. At the level of individual

calves (not shaded part right upper corner) and at herd level (shaded part left lower part).

Presented values are Spearman-correlation-coefficients (p-values).

E. coli Corona virus Cl. perfringens C. parvum Rota virus

E. coli - 0.15 (0.03)

Corona 0.27 (0.004) - 0.10 (0.036)

Cl. perfringens -

C. parvum 0.26 (0.007) 0.17 (0.080) - 0.17 (0.001)

Rota 0.23 (0.017) 0.20 (0.040) -

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The combination in which different intestinal pathogens were found simultaneously is

represented in table 4. At the level of individual calves, E. coli was significantly more present in

relation with Cl. perfingrens. The same relation was found for the presence of Corona and Rota

virus in calves with C. parvum.

Also at herd level, a relation was found between the presences of Corona or Rota virus in

combination with C. parvum. Also E. coli and Corona virus seemed to be present simultaneously in

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frequent cases. Finally Cl. perfringens was found significantly more frequent in calves, whereby

also Rota virus was present.

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Relation of intestinal pathogens and the faeces consistencies

From this analysis, it appeared there was no relation between sex and age with faecal

consistencies. The same was found for type of housing and animal symptoms like tenting of the

skin and heart beat.

Calves with a C. parvum infection had an increased risk (OR=2.1) for custard like

consistent faeces. Besides, there was an indication that calves with a Rota virus infection, also had

custard-like consistent faeces (OR=1.8) and that calves that got bulk milk in stead of artificial

prepared milk, had less frequent custard-like consistent faeces (OR=0.5) (table 5).

Table 5. Relation of found intestinal pathogens in 424 calves in 108 dairy herds and the presence

of custard like consistent and watery faeces. Also some clinical and management factors were

taken into account (OR=Odds Ratio).

Custard consistent-like

faeces OR 95% Confidence interval. p-value

Rota virus 1.7 0.9 3.2 0.11

E.coli 2.2 0.5 10.6 0.32

C. parvum 2.1 1.2 3.7 0.02

Cl. perfringens 1.0 0.6 1.6 0.99

Under temp. (T < 380C 0.6 0.3 1.3 0.22

Fever (Temp > 39.50C) 1.7 0.4 6.7 0.46

Treated calves/in treatment for diarrhoea 1.2 0.7 2.0 0.50

Artificial

prepared milk Reference

Bulk milk 0.4 0.2 0.9 0.04

Cow milk 0.7 0.4 1.2 0.19

Milk supply at the

moment of sampling

Electrolytes 1.4 0.5 3.9 0.58

Colostrums 0.6 0.3 1.3 0.21

Watery-like faeces OR 95% Confidence interval p-value

Rota virus 2.2 1.1 4.6 0.03

E.coli 6.6 1.3 34.5 0.03

C. parvum 6.2 3.3 11.6 0.00

Cl. perfringens 2.1 1.1 3.8 0.02

Under temp.(T. < 380C) 0.2 0.1 0.8 0.02

Fever (T. > 39.50C) 4.0 1.0 16.0 0.05

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Treated calves/in treatment for diarrhoea 2.0 1.1 3.9 0.03

Artificial prepared

milk Reference

Bulk milk 0.4 0.1 1.2 0.11

Cow milk 0.9 0.4 1.8 0.70

Milk supply at the

moment of

sampling

Electrolytes 4.2 1.3 13.1 0.01

Colostrums 0.9 0.3 2.6 0.88

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All intestinal pathogens except Corona virus showed a relation with watery faeces. The

highest effect was the presence of C. parvum (OR=6.2) and E. coli (OR=6.6). It also appeared that

calves with fever, had more frequent watery faeces (OR=4.0), while calves with under temperature

had on the other hand less frequent watery faeces (OR = 0.2). In addition to this, it was found that

calves that were fed electrolytes at the moment of sampling, had 4.2 times more frequently

diarrhoea than calves fed artificial prepared milk.

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Factors associated with E. coli

Most important factors associated with the presence of E. coli in the faeces (table 6):

1. presence of Corona virus in calves (’faeces) between 1 – 21 days at the herd;

2. under temperature, There was an indication that calves with a low body temp., were infected

with E. coli more frequently than calves with a higher body temp.;

3. age, there was an indication that calves between 1-7 days of age had more frequent E. coli

infections than older calves;

Corrected for the above mentioned factors, there was an indication that calves with an E. coli

infection had more frequent watery faeces.

Table 6. Factors associated with the presence of E. coli in the calves’ faeces (n=424) at dairy

herds (n=108) in The Netherlands (OR=Odds Ratio).

OR 95% Confidence interval p-value Corona at the herd 3.7 1.0 13.6 0.05 Under temp. 3.9 1.0 15.9 0.06 Age week 1 6.6 0.8 55.6 0.08 week 2 Reference week 3 3.7 0.3 43.9 0.30 Consistencies of faeces Normal Reference Custard-like 1.9 0.4 9.2 0.44 Watery 4.0 0.9 18.1 0.07 274

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Factors associated with Corona virus

The most important factors associated with the presence of Corona virus in the faeces (table 7):

1. presence of E. coli in calves (’ faeces) between 1 – 21 days at the herd;

2. presence of C. parvum in calves (’faeces) between 1 – 21 days at the herd. An indication for

a higher frequency of a Corona virus infection in calves was found in case of presence of C.

parvum at the herd;

3. frequent cleaning of the group housing.

Corrected for factors mentioned above, there was an indication that calves with a Corona virus

infection had more frequent custard-like consistent and watery faeces.

Table 7. Factors associated with Corona virus in the calves’ faeces (n=424) at dairy herds (n=108)

in The Netherlands (OR=Odds Ratio).

OR 95% confidence interval p-value presence of E. coli 4.6 1.1 18.6 0.03 indication for C. parvum 8.4 0.9 77.9 0.06

Yes Reference housing cleaned after group removal No 8.2 2.2 30.0 0.00 faeces consistencies Normal Reference Custard like 5.1 0.9 28.0 0.06 Watery 4.4 0.8 25.5 0.10

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Factors associated with C. parvum in the faeces:

Most important factors associated with the presence of C. parvum (table 8):

1. presence of Rota virus in calves (’ faeces) at the herd.

2. treatment. In calves which were treated or were in treatment at the moment of herd visiting,

C. parvum was more frequent found.

3. electrolytes supply. Calves that were supplied electrolytes in stead of milk at the moment of

herd visiting, C. parvum was more frequent found in faeces;

4. cleaning of the buckets with hot water. An indication was found that in herds that claened the

buckets with hot water, C. parvum was less frequent present than in herds that claened the

buckets with cold water.

5. age. In calves in their 2nd week of life C. parvum was found more frequent in the faeces than

in calves’ faeces in 1st and 3rd week of age;

Corrected for factors mentioned above calves with watery faeces had more frequent C. parvum

than calves with faeces of normal or custard-like consistency.

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Table 8. Factors associated with the presence of C. parvum in the calves’ faeces (n=424) at dairy

herds (n=108) in The Netherlands (OR=Odds Ratio).

OR 95% Confidence interval p-value presence of Rota at the herd 2,4 1.2 4.9 0.02 electrolyte supply Milk Reference Electrolytes 6.3 1.7 22.3 0,01 calf treated/under treatment 2,3 1.1 5.0 0.03 cleaning buckets cold water Reference hot water 0.5 0.2 1.1 0,09 age of calf Week 1 0.1 0.1 0.3 0,00 Week 2 Reference Week 3 0.2 0.1 0.5 0,00 faeces consistencies Normal Reference custard-like 1.7 0.8 3.6 0,13 Watery 6.5 3.1 14.0 0,00

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Factors associated with Rota virus

Most important factors associated with the presence of Rotavirus in the faeces (table 9):

1. the number of calves present at moment of herd visiting. An indication was found that the

more calves between 1 - 21 days of age were present, the higher the risk of Rota virus

infections;

2. presence of calves with diarrhoea. When others calves with diarrhoea were present, the risk

of presence of a Rota virus infection increased;

3. age. In calves in their 2nd week of life Rota virus was found more frequent in the faeces than

in calves’ faeces 1st and 3rd week of age;

Corrected for factors mentioned above, an indication was found that calves with watery faeces had

more frequent Rota virus than calves with faeces of normal or custard-like consistency.

Table 9. Factors associated with Rota virus in the calves’ faeces (n=424) at dairy herds (n=108) in

The Netherlands (OR=Odds Ratio).

OR 95% confidence interval p-value No. of calves present 1,1 1.0 1.3 0.09

No Reference Calves with diarrhoea present Yes 2.2 1.1 4.6 0.03 Calves’ ages Week 1 0.6 0.3 1.1 0.07 Week 2 Reference Week 3 0.4 0.2 0.9 0.02 Consistencies of faeces Normal Reference

Custard-consistency 1.8 0.9 3.7 0.11

Watery 2.6 1.2 5.4 0.01 319

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Factors associated with Cl. perfringens

Most important factors associated with the presence of Cl. perfingens in the faeces (table 10):

1. age. Especially in calves during their 1st week of life Cl. perfringens was found more

frequently.

2. consistencies of faeces. For the presence of watery faeces and Cl. perfringens a strong

relation was found.

Table 10. Factors associated with Cl. perfringens in the calves’ faeces (n=424) at dairy herds

(n=108) in The Netherlands (OR=Odds Ratio).

OR 95% Confidence interval p-value Consistencies of faeces Normal Reference Custard-like 1.0 0.6 1.8 0.96 Watery-like 2.3 1.2 4.4 0.02 Calves’ ages Week 1 Reference Week 2 0.5 0.3 0.8 0.01 Week 3 0.3 0.2 0.6 0.00

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Discussion

The objective of this prevalence investigation was to acquire insight into the prevalence of

diarrhoea and of 5 intestinal pathogens in calves at dairy herds in The Netherlands and related

management factors.

The results showed that E. coli and Corona virus infections in calves between1 - 21 days at

dairy herds were present in low frequency. Remarkable was that those infections were in practically

all cases present in only 1 calf of a herd. C. parvum, Cl perfringens and Rota virus were found

much more frequently and in those cases the infection concerned more calves within the same

herd.

This investigation could only be performed with the help of dairy farmers. Therefore 309 dairy

farmers in The Netherlands were approached. In spite of the large number (n=141) of the farmers

that were prepared to cooperate with the proposed investigation for sure, at the moment of the

suggested herd visit, these farmers had no or only 1 calf present within the preconditioned age of

1-21 days of age. A small number of farmers approached (n=38) had no interest for this

investigation. A reason for lack of interest could be a lack of interest for all kind of investigations,

or related to a supposed irrelevance, because the (practically total) absence of clinical problems

with diarrhoea in young calves in their herds. With a small number of selected farmers (n=9) no

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contact could be made, despite several attempts at different moments of the day. The extent of the

influence, of this undesired selection, on the results of this investigation is hard to determine, but

is considered to be limited. Because of the conditional criterion of 2 calves within a stage of age of

1-21 days at least, there was a slight bias to larger dairy herds. However, in the analyses per

intestinal pathogen, hardly any influence on herd size could be demonstrated; only for Rota virus

an effect for the number of calves present in a herd was estimated. Based on ad random selection

of the herds, a distribution of the visited herds in The Netherlands was normal; in the investigators’

opinion there are no reasons to suppose that the prevalence and risk factors of intestinal

pathogens of the investigated 108 herds are not representative for the intestinal pathogens of

calves within the first three weeks after birth in The Netherlands.

In this investigation not normal faeces was found in 43% of the calves. Most frequent abnormal

faeces were found during the 2nd week after birth (51.6%). Custard consistency-like faeces were

somewhat more often diagnosed than watery-like faeces, but also concerning this point disparities

were found per week of age. The effect of intestinal pathogens on the faeces consistency was

obvious for watery-like faeces, but less obvious for custard consistency-like faeces. Only the

presence of C. parvum had a statistically association with custard consistency-like faeces.

Remarkable was the association with custard consistency-like faeces and the supply of bulk milk in

stead of artificial- prepared milk. The OR of 0.4 indicates that calves that were offered bulk milk

produce 1/0.4 = 2.5 time more frequent normal faeces than calves that were offered artificially

prepared milk. Bulk milk might be more stable for the composition than artificial-prepared milk and

it composed (especially) some immunoglobulin, which has protectional qualities, that is to say in

(very) low concentration.

With the exception of Corona virus, the other faecal pathogens had a significant correlation

with watery-like faeces. The results of this investigation have shown clearly, that the presence of

watery-like faeces is related to an infection with one of the investigated pathogens. In a

quantitative way, this effect was largest for E. coli and C. parvum. Besides, associations were

found with body temperature, (diarrhoea) treatment and origin of milk supplied. The opposite

relation was found for under temperature and fever in combination with watery-like faeces; this

was physiologically seen not remarkable. In fact, under temperature is an indication for a

dysfunction of the thermoregulation. Sufficient body fluid is in that case a first condition and

diarrhoea can hardly be present in dehydrated calves. This is different for a reaction of fever. Fever

has a function to eliminate an infection by increased body temperature and activation of the

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immunoreactions and above that an increased intestinal mobility has a function to eliminate

intestinal infections.

The estimated prevalence at animal and herd level are relatively low for the agents E. coli and

Coronavirus (respectively 2.6% and 3.9%), but these prevalence are comparable with

investigations in UK, Ireland, Spain and Switzerland (Snodgrass et al., 1986; Fagan et al., 1995;

Fuente et al., 1998; Lugingbühl et al., 2005) and the results of an investigation in The Netherlands

at post mortal calves Van Maanen et al. (2006). Lugingbühl et al. (2005) estimated in a prevalence

study in (mainly diarrhoeic) suckling calves above all C. parvum (43%) and Rota virus (46%) and

in low frequency E. coli and Corona virus. Van Maanen et al. (2006) estimated in their investigation

of neonatal diarrhoeic calves, in only 6% of the calves an E. coli infection. Because E. coli is mainly

seen in the first week and may result in quick death, it is possible that the prevalence of E. coli is

somewhat underestimated in our study. However, based on information from this survey, no

significant association could be found between an E. coli infection and dead of neonatal calves in

dairy herds. Beside that, the value of the OR estimation was just smaller than 1.

In publications mentioned Rota virus and C. parvum (prevalence respectively 17.7% and

27.8%) are considered to be “major pathogens”; this is in line with the findings in our

investigation. The estimated high prevalence of Cl. perfingens at animal (54%) and herd level

(85%) is a replacement for the opinion that this genus of bacteria has to be considered as an

intestinal commensal. In this investigation, no further differentiation has been made between the

different types of C. perfringens and the capability to produce toxins. An additional study, based on

e.g. a cohort design, whereby additionally further typing of the Cl. Perfringrens spp. and the

production of toxins is involved, may provide more information about a possible pathogenicity of

this agent in the aetiology of (neonatal) diarrhoea in calves during the first months.

Concerning the risk factors at herd level, E. coli was significantly more frequently found in

the first week after birth and in calves with under temperature. This finding was in line with

literature (e.g. Radostits et al., 2006). More remarkable was the higher prevalence of E. coli

simultaneously with the presence Corona virus. This might be explained by considering E. coli

infections to be secondarily to a primarily Corona virus infection. This association was also found by

analysis of the risk factors of Corona virus. Beside E. coli, also the presence of C. parvum was

found to be a risk factor for a Corona virus infection. This is remarkable, because in literature C.

parvum is especially associated with Rota virus (Waltner-Toews et al., 1986; Bellinzoni et al.,

1990; Garcia et al., 2000; Joachim et al., 2003). Both with these agents and with C. parvum the

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influence of a good hygiene of the housing was found, a well known risk factor for what the advice

for individual housing and isolation of diarrhoeic calves is based on (Radostits et al., 2006).

The positive association between C. parvum and both diarrhoea treatment and supply of

electrolytes, substitutes the strong relationship between C. parvum infection and the presence of

clinical symptoms of diarrhoea. Apart from that, a positive association was found for C. parvum

with the presence of Rota virus in dairy herds. Based on correlation coefficients (be aware: it

concerns 1 to 1 relations, without corrections for possible confounding factors) the presence of C.

parvum was also associated with Corona virus and Cl. perfringens infections. Based on these data

C. parvum seems to play an important role in the aetiology of clinical diarrhoea problems in calves

from 1-21 days and the treatment of diarrhoea as a herd problem has to include the prevention

and treatment of C. parvum at least. The significantly more frequent presence of C. parvum in the

2nd week of life is in accordance with the literature, whereby the highest excretion is mentioned

between D9 - D14 also (Radostits et al., 2006).

The risk of presence of Rota virus was significant increased during the 2nd week of life, in

calves with watery-like faeces and the presence of other diarrhoeic calves and a statistically

significant relation was found for Rota virus and the presence of more calves in a herd. This

underlines the infectious character of Rota virus and this is also in line with literature (Radostits et

al., 2006).

Concerning the risk factors founded for diarrhoea and the presence of Cl. Perfingens,

comparable studies are not known. Based on correlations, only increased trends and significant

relations were found with respectively C. parvum and Rota virus (Table 4). Although in the

multivariable analysis these relations were not significant anymore (Table 10). These apparent

connections were too much influenced by confounders like age and consistency of the faeces. This

seems to justify the conclusion that a Cl. perfringens infection is not related to other intestinal

pathogens and has without further typing and/or proven toxin production of this bacterium, to be

considered as an intestinal commensal.

Conclusions

At 108 dairy herds, with in totally 424 calves within an age of 1-21 days, a cross-sectional

study with (including investigation of faecal samples of individual calves and a survey) was

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performed, whereby abnormal faeces in 53% of the calves were estimated. A C. parvum infection

was the most important factor for both custard consistency-like and watery-like faeces.

E. coli and Corona virus infections were found in a more incidentally way, while Cl. perfringens

infections were found in more than 50% of the investigated calves. Based on the results Cl.

perfringens can not be considered as a intestinal pathogen, but more as an intestinal commensal.

C. parvum and Rota infections virus were present in respectively 27.8% and 17.7% of the calves.

Related to C. parvum, Corona and Rota virus some herd factors (hygiene measurements, pressure

of infection) were statistically associated.

Acknowledgements

This investigation was a DAHS Project: 8071009 diarrhoea in calves at dairy herds. The

cooperation of all dairy farmers and the personal of the DAHS laboratory in this study were greatly

appreciated. This study was funded by Schering Plough Animal Health (Maarssen, The

Netherlands).

Acknowledgements

This investigation was as DAHS Project: 8071009 diarrhoea in calves at dairy herds. The

cooperation of all dairy farmers and the personal of the DAHS laboratory in this study were greatly

appreciated. This study was funded by Schering Plough Animal Health (Maarssen, The

Netherlands).

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References

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Dairy Science 68, 229-256.

2. Bellinzoni RC, Blackhall J, Terzolo HR, Moreira AR, Auza N, Mattion N, Micheo GL, La Torre JL,

Scodeller EA. 1990. Microbiology of diarrhoea in young beef and dairy calves in Argentina.

Rev Argent Microbiol. 22:130-6.

3. Björkman, C., Svensson, C., Christensson, B. en Verdier, K. de. 2003. Acta vet. Scand. 44;

145-152.

4. Curtis, C.R., Erb, H.N., Scarlett, J.M. en White, M.E.. 1988. Path model of herd level risk

factors for calfhood morbidity and mortality in New York Holsteins herds. J. Dairy Sc. 78:

2709-25.

5. Fagan, J.G., Dwyer, P.J., Quinlan, J.G., 1995. Factors that may affect the occurrence of

enteropathogens in the faeces of diarrhoeic calves in Ireland. Irish Vet. J. 48: 17-21.

6. Fledderus A, Woutersen-van Nijnanten FM, Holzhauer C, van den Ingh TS, van Dijk JE,

Nijkamp FP. 1992 Hyperreactivity to histamine in the jejunum of veal calves with diarrhoea.

Vet Rec. 130: 207-278.

7. Fuente, de la, R., Garcia, A., Ruiz-Santa-Quiteria, J.A., Luzon, M., Cid, D., Garcia, S., Orden,

J.A., Gomez-Bautista, M. 1998. Proportional morbidity rates of enteropathogens among

diarrheic dairy calves in central Spain. Prev Vet Med. 36: 145-152

8. García, A., Ruiz-Santa-Quiteria, J.A., Orden, J.A., Cid, D., Sanz, R., Gómez-Bautista, M. en

La Fuente, R. de. 2000. Rotavirus and concurrent infections with other enteropathogens in

neonatal diarrheic dairy calves in Spain. Comparative Immunology, Microbiology & Infectious

diseases. 23;175-183.

9. Geurden, T., Claerebout, E. en Vercruysse, J. 2005. Protozoaire infecties als oorzaak van

diarree bij jonge kalveren. Tijdschrift voor diergeneeskunde 130:734-737.

10. Graaf, D.C., de, Vanopdenbosch, E., Ortega-Mora, L.M., Abbassi, H. en Peeters, J.E.. 1999. A

review of the importance of cryptosporidiosis in farm animals . Int. J. Parasitol; 29: 1269-

1287.

11. Joachim, A., Krull, T., Schwarzkopf, J., Daugschies, A. 2003. Prevalence and control of

bovine cryptosporidiosis in German dairy herds. Vet Parasitol. 112(4):277-88.

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12. Kaneene, J.B. and Hurd, H.S., 1990. The National Animal Health monitoring system in

Michigan. I. Design, data frequencies of selected dairy cattle diseases. Prev. Vet. Med. 8:

103-114.

13. Lebrun, M. Filée, P., Mousset, B., Desmecht, D., Gelleni, M., Mainil, J.G. en Linden, A. 2007.

The expression of Clostridium perfingensconsensus beta2 toxin is associated with bovine

enterotoxaenemie syndrome. Vet. Microbiology 120, 151-157.

14. Lorenz, I., 2006. Diarrhoea of the young calf: an update. World Buiatrics Nice.

15. Lüginbühl, A., Reitt, K., Metzler, A., Kollbrunner, M., Corboz, L., Deplazes, P. 2005. Field

study of the prevalence and diagnosis of diarrhea-causing agents in the newborn calf in a

Swiss veterinary practice area. Schweiz Arch Tierheilkd. 2005. 147: 245-52.

16. Maanen, van C., Mars, M.H., Meulen, van der, A.M., Sande, van de H., Blok, H.A., Reusken,

C.B.E.M., 2006. Detection of enteropathogens involved in calf neonatal diarrhoea: validation

of commercially available ELISA’s and lateral flow immunoassays as compared with reference

methods. Proceedings 7th International Congress of Veterinary Virology, September 2006, p.

288.

17. Muskens, J., Veldhorst, G.J., Snoep, J.J., Vos, J. 2007. High mortality in a herd with signs of

jejunal hemorrhage syndrome Tijdschr Diergeneeskd. 132:116-9.

18. Nussbaum, D.J., Salord, J.R. en Rimmele, D.D. 1999. Evaluation of quantitative latex

agglutination for detection of Cryptosporidium parvum, E. coli K99, and rotavirus in calf

feces. J. Vet. Diagn. Invest. 11:314-318.

19. Olson, M.E., Gob, J., Phillips, M., Guselle, N. en McAllister, T.A.. 1999. Giardia cyst and

Cryptosporidium parvum oocysts survival in water, soil and cattle feces. Journal of

Environmental Quality 28, 1991-1996.

20. E. Perez, Noordhuizen, J.P.T.M., Wuyckhuise, L.A. van en Stassen E.N. 1990. Management

factors related to calf morbidity and mortality rates. Livest. Prod. Sc. 25: 79-93.

21. Radostits, O. M., Gay, C. C., Hinchcliff, K.W. 2006. Veterinary Medicine, A Textbook of the

Diseases of Cattle, Horses, Sheep, Pigs and Goats.

22. Snodgrass, D.R., Terzolo, H.R., Sherwood, D., Campbell, I., Menzies, J.D., Synge, B.A. 1986.

Aetiology of diarrhoea in young calves. Vet Rec. 119:31-4.

23. Subnel, A.P.J., Boxem, T., Meijer R.G.M. en Zom, R.L.G. 1994.Voeding van melkvee en

jongvee in de praktijk. Proefstation voor Rundveehouderij, Schapenhouderij en

Paardenhouderij (PR), Lelystad, 145 pp.

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24. Tzipori, S. 1981.The aetiology and diagnosis of calf diarrhoea. Vet. Rec. 108.510-514.

25. Tzipori, S. 1985. The relative importance of enteric pathogens affecting neonates of domestic

animals. Adv. Vet. Sci. Comp. Med. 29:103-206.

26. Waltner-Toews, D., Martin, S.W., Meek, A.H.. 1986a. An epidemiological study of selected

calf pathogens on Holstein dairy farms in south-western Ontario. Can J Vet Res. 50: 307-13.

27. Waltner-Toews, D., Martin, S.W. en Meek, A.H.. 1986b. Dairy calf management, morbidity

and mortality in Ontario Holstein herds. II. Ages and seasonal patterns. Prev. Vet. Med. 4:

125-35.

28. Wells, S.J., Dargatz, D.A. en Ott, S.L.. 1996. Factors associated with mortality to 21 days of

life in dairy heifers in the U.S. Prev. Vet. Med. 29: 1-19.

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Survey about the management of calves from 1-21 days Project 8071009 diarrhoea in calves in dairy herds General herd information Name herd:………………………………………………………………………………… UBN herd:…………………………………………………………………………………. Date visit:………………………………………………………………………………..

A. How many cows have calved during the last 3 weeks? No.:……………………………………………….

B. How many heifers have calved during the last 3 weeks?

No.:………………………………………………. C. How many calves are at this moment in the herd (0-21days)?

No.:……………………………………………….

D. What is the total no. of calves that died the last 3 weeks? No.:……………………………………………….

E. How many of the calves have been treated (0-21days)? No.:………………………………………………

F. Which race are the animals in your herd at the moment? Race:…………………………………………………..

G. What kind of herd management is performed?

Traditional, Biological, Ecological Housing 1. How the calves are housed (0 – 21 days)?

a. individual in single boxes until ……….……..days; b. individual in straw boxes until…...…………..days; c. individual in a calf hut outside unt.…………..days; d. in group housing from…..……………………days; e. near the dairy cows, during:…...……………..days;

2. At which age are the calves transferred to groups housing?

..……………..days.

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3. What is the first time colostrums is offered to the calves? a. Always within1- 2 hours; b. Always within 2- 6 hours; c. Calf stays with the cow; d. Calving at night, feeding in the morning; e. Different, namely: ……………………………………………………………………..

4. How many colostrums is fed to new born calves? (more answers possible)

a. immediately (< 1 hour) 1 - 1,5 litres b. first day ≥4 litres; c. second day ≥6 litres; d. ad libitum;

5. When (day) starts the feeding of artificial prepared milk?

a. Day 2; b. Day 3; c. Later, namely: … d. Never, the calves get cow milk

Treatment of and prevention for sick calves) 6. Does the herd apply preventive vaccinations at the dairy cows and/or heifers related to

calf diarrhoea? a. no, never; b. yes, always with the therapeuticum:…………………………………………………. c. yes, sometimes in case of problems, de last time was (year) ………………………….

7. Do you use any preventive medicines at calves within 1- 21 days of age?

a. Yes, why;…………………………………………………………….. b. Yes, artificial prepared colostrums (name); c. Which medicines do you use? ……………………………………….. d. No;

8. How are diarrhoeic calves treated (until 21 days)? (More answers possible)

a. Antibiotic containing powder (name: ………………….…………………………..); b. electrolytes; c. antibiotics, by injection; d. oral medicines (name: …………………………………..); e. different, namely.:………………………………………

Nursing of the calves 9. Does the person, who takes care of the calves, use clothes specific belonging to the calves?

a. yes, always; b. yes, somtimes; c. no.

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10. Who treats the calves normally? (more answers possible) a. the farmer’s wife b. the farmer c. other personal in the herd/stagier d. others, namely:

Cleaning 11. Cleaning and disinfection of the boxes:

a. every time after removal of the calf to group housing--> clean straw; b. every time after removal of the calf to group housing, cleaning, disinfection and

clean straw; c. as b, but different boxes simultaneously; d. 1-2x per year; e. never.

12. Cleaning of the buckets / bottles:

a. every time after feeding of the calf, the buckets / bottles cleaned with hot/cold water;

b. the same as a., but cleaning/disinfection after the group; c. No cleaning/disinfection buckets/bottles after feeding; d. All materials cleaned/disinfected hot/cold at the end; e. All cleaning with a cleaning-/ disinfection product, namely: ……….. f. Different;

Bull calves 13. By whom are the bull calves normally delivered for the fattening industry?

a. always the same dealer b. not always the same dealer c. others, namely;

14. How are the bull calves normally delivered to the dealer?

a. via a special exit; b. via the (central)herd entrance (where every visitor comes in); c. are delivered at the exit; d. these bull calves are kept separately in a calf hut, or at another different place in the

herd; e. different, namely:

Other affairs 15. Is there any peak of calf birth within the herd (more than 50% within a period of 3 month)

a. Autumn; b. Spring; c. Different peak; d. Not any peak.

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