PJZ_57_4_1855-1862

INTRODUCTION

Diarrhea represents the primary cause of both high morbidity and mortality among young animals, resulting in substantial economic losses in the global livestock industry (Yadegari et al., 2019; Shrivastava et al., 2022). Diarrhea leads to deficit of health and drop in production potential of animals (Koirala and Bhandari, 2019). According to the National Animal Health Monitoring System, approximately 57% of calf deaths at the weaning stage can be attributed to diarrhea (Cho and Yoon, 2014). Small ruminants play a vital role in the agricultural systems of developing countries, both economically and ecologically (Devendra, 2005). The mortality rate for lambs typically falls within the range of 10 to 25%, while estimates for goat kid mortality range from 11.5 to 37% (Thiruvenkadan and Karunanithi, 2007).

The causes of diarrhea in sheep and goats encompass both non-infectious and infectious factors (Abdou et al., 2021). Among bacterial pathogens, enterotoxigenic E. coli (ETEC) and Salmonella hold significant economic importance (Achá et al., 2004). Although the presumed cause is typically attributed to specific bacteria like E. coli, investigations into diarrhea among both young kids and adult goats have consistently been unable to identify specific causes in the majority of instances (Gavin et al., 2018). In both cattle and dairy goats, research has demonstrated variations in the expression patterns of different virulence genes in E. coli, depending on the host’s age, season and geographic location (Dwell et al., 2008; Fernández et al., 2009). E. coli typically induces watery diarrhea and weakness in newborn calves aged 1–4 days. Usually, death often ensues within 24 h as a result of severe dehydration (Cho et al., 2010). Additionally, stress has been identified as a factor that can lead to an elevated prevalence of specific virulent strains (Bach et al., 2004; Ndegwa et al., 2020). Salmonella infection stands as a prevalent cause of diarrheal illness in both sheep and goat flocks (Shabana et al., 2017). The extent of Salmonella infection’s impact varies, contingent on bacterial factors such as serovar, virulence, and antimicrobial susceptibility. It’s worth noting that the global count of identified Salmonella serovars exceeds 2600, with new serovars emerging annually (Jajere, 2019).

Understanding the epidemiology, prevalence, and virulence factors of these pathogens in specific regions is crucial for effective disease management and prevention strategies. The current research was conducted to study the prevalence of E. coli and Salmonella in diarrheic goats in Bahawalpur, Pakistan (Fig. 1).

MATERIALS AND METHODS

A total of 200 goats suffering from clinical signs of diarrhea (irrespective of breed and sex) up to two months of age were included in this study. The fecal samples were collected directly from the diarrheic goats and the data was recorded using a pre-tested form termed as Data Capture form (DCF). The parameters in DCF included age, breed, sex, duration of diarrhea, fecal scoring, season, stagnant water, colostrum feeding, environmental condition, and body condition.

Each animal’s rectum was used to collect approximately 5 g of feces. DNA from colonies of positive samples were extracted using bacterial DNA extraction kit (WizPrepTM gDNA cell/tissue kit, Korea) and confirmed through PCR (Haq et al., 2017) by using new primers.

Isolation and purification of E. coli and Salmonella

Bacterial pathogens like E. coli and Salmonella were identified and isolated by culturing (Kar et al., 2017; Haq et al., 2017). The samples were mixed with 5mL phosphate buffer saline solution in test tubes, followed by inoculation on Nutrient Agar were further streaked on MaCconkey agar and EMB agar and incubated further at 37℃ aerobically for 24 h. Only pink colonies from MaCconkey agar, and green metallic sheen colonies from EMB agar were chosen for E. coli. These colonies were further purified by multiple streaking methods on the MacConkey agar and EMB agar and further processed for identification and confirmation of E. coli.

For isolation and purification of Salmonella, bacterial cultures were streaked on to nutrient agar, yielding translucent circular Salmonella colonies after 24 h and then streaked onto Salmonella-shigella agar (SSA), incubated at 37°C for 24 h, and processed to purify the bacteria by multiple streaking method.

Pure colonies were proceeded for identification of E. coli by observing macroscopic, microscopic and biochemical characters. It was followed by motility test using hanging drop method (Cheesbrough, 2009).

The isolates that were Gram negative after the gram staining were then subjected for the biochemical tests which include different tests, MR test, VP test, indole test, catalase test, oxidase test, citrate utilization test and urease test and H2S production tests (Reiner, 2010; Koneman, 2016).

Molecular diagnosis of bacteria

For molecular diagnosis of bacteria positive fecal samples were processed for detection of E. coli and Salmonella DNA, using primer sequence specific for inaA gene and F5 gene (Tables I and II).

Table I. Primer sets used for characterization of E. coli and Salmonella.

Gene	Primer sequence 5’ →3’	Length of PCR product (bp)
invA	F TTGTTACGGCTATTTTGACCA	521
	R CTGACTGCTACCTTGCTGATG
F5	F TATTATCTTAGGTGGTATGG	314
	R GGTATCCTTTAGCAGCAGTATTTC

Table II. Biochemical characteristics of Salmonella and E. coli.

Biochemical test	Salmonella	E. coli
Gram staining	Negative	Negative
Motility	Positive	Positive
Catalase test	Positive	Positive
Oxidase test	Negative	Negative
Indole test	Negative	Positive
Methyl red test	Positive	Positive
Citrate utilization test	Positive	Negative
VogesProskauer test	Negative	Negative

For genomic DNA extraction of Gram-negative bacteria fresh culture of isolates was taken from MaCconkey agar plates and transferred into micro centrifuge tube (1.5ml) containing normal saline from fecal samples using genomic DNA extraction Kit (GeneAll®, Exgene™ Catalogue No. 105-101).

PCR recipe was arranged in an absolute volume of 20μl containing of 10μl of TOPreal™ qPCR 2x PreMIX, 2μl of DNA sample and 1μmol of separately primer. Reaction was cycled 35 times afterwards first denaturation at 95°C for 5 min with denaturation at 95°C, annealing at 60°C and extension phase at 72°C, each phase was given 45 seconds, last elongation at 72°C for 10 min was done. A negative control was involved in each PCR run. The PCR yields were detected on agarose gel (2%)for positive bands against a 100bp molecular ladder. Amplified products were visualized using UV Transilluminator.

Statistical analysis

The data were statistically analyzed using SPSS 25.0 version. Analysis of variance (ANOVA) was used in case of more than two variables within group while Student’s t-test was employed for the two groups.

RESULTS

Prevalance of E. coli and Salmonella in diarrhoeic goats

Out of 200 samples 47 goats were positive for E. coli and 35 goats were positive for Salmonella infection after initial identification on basis of microscopic examination and biochemical profile. The PCR analysis confirmed the presence of Salmonella by 521bp amplified band of invA gene and E. coli by amplified band of K99 gene.

The results revealed that bacterial prevalence was 43.5% (97/200). Among which E. coli prevalence was 23.5% and Salmonella prevalence was 17.5% in Bahawalpur district, Punjab, Pakistan.

Age

The goats were examined across three age groups: G1 (0-15 days), G2 (16-30 days), and G3 (>30 days). Among these groups, 18.60% goats in G1, 22.22% goats G2, and 13.83% goats in G3 were tested positive for Salmonella (Table III). The statistical analysis indicated that age was a significant factor (P>0.05) in relation to Salmonella infection. In case of E. coli infection, maximum positive cases were recorded in G1 (34.88%) while in G2 and G3 the positive cases were 20.63% and 20.21%, respectively (Table III). In the present study, age factor was significant (P<0.05): Salmonella infection was found to be most infected G2 while G1 was found to be most infected age group with E. coli.

Breed

The results showed that Desi breed has the maximum rate (21.21%) of infection followed by Beetal (19.35%), Teddy (17.78) and Rajanpuri breeds (13.33) (Table III). The results were non-significant (P>0.05) for Salmonella infection causing diarrhea.

In case of E. coli, the results showed that Desi breed showed maximum (29.41%) positive cases followed by Teddy (26.09%) and Beetal (24.59%). The Rajanpuri breed had the least infected cases in terms of percentage (16.95%). The variations on the basis of breed were found to be significant (p<0.05). In both cases, Desi breed was most infected.

Gender/Sex

Regarding sex, male goats showed more prevalence in both infections: Salmonella 65.71% and E. coli 31.25% (Table III). Infections in female were 7.27% in the case of Salmonella while 13.64% in the case of E. coli. The results were significant (P<0.05) in both infections.

Table III. Prevalence of Salmonella and Escherichia coli infection in diarrheic goats kids.

Parameters	Salmonella		E. coli
Parameters	Positive/ total (%)	P value	Positive	P value
Age
0-15 days	8/43 (18.60 %)	0.038	15/43 (34.88 %)	0.013
16-30days	14/63 (22.22%)		13/63 (20.63 %)
>30days	13/63 (13.83%)		19/94 (20.21 %)
Breed
Teddy	8/45 (17.78 %)	0.756	12/46 (26.09 %)	0.001
Beetal	12/62 (19.35%)		15/61 (24.59 %)
Rajanpuri	8/60 (13.33 %)		10/59 (16.95 %)
Desi	7/33 (21.21 %)		10/34 (29.41 %)
Sex
Male	23/35 (65.71%)	0.001	35/112 (31.25%)	0.004
Female	12/165 (7.27%)	0.001	12/88 (13.64 %)	0.004
Season
Winter	7/17 (41.18 %)	0.073	12/46 (26.09 %)	0.001
Spring	6/46 (13.04 %)		10/59 (16.95 %)
Summer	13/68 (19.12%)		15/61 (24.59 %)
Autum	9/69 (13.04 %)		10/34 (29.41 %)
Colostrum feeding
Yes	26/128(20.3%)	0.016	19/102 (18.63%)	0.097
No	9/72(12.50 %)	0.016	28/98 (28.57 %)	0.097
Duration of diarrhea
1 Day	8/57 (14.04 %)	0.066	22/84 (26.19 %)	0.063
2 Day	13/64 (20.31%)		14/71 (19.72 %)
3 or >3 days	14/79 (17.72%)		11/45 (24.44 %)
Body condition
Fatty	4/51 (7.84 %)	0.579	8/43 (18.60 %)	0.027
Emaciated	20/82 (24.39%)		30/94 (31.91%)
Good	11/67 (16.42%)		9/63 (14.29 %)
Environmental condition
Good	8/57 (14.04 %)	0.069	9/71 (12.68 %)	0.037
Fair	11/61 (18.03%)		22/47 (46.81%)
Poor	16/82 (19.51%)		16/82 (19.51%)
Stagnant water
Yes	23/118 (19.4%)	0.037	34/108 (31.48%)	0.004
No	12/82 (14.63%)	0.037	13/91 (14.13 %)	0.004
Fecal scoring
1 (Normal feces)	4/21 (19.05%)	0.047	4/18 (22.22 %)	0.003
2 (Semi solid feces)	12/73 (16.44%)		10/46 (21.74%)
3(Dysentery)	6/49 (12.24 %)		3/35 (8.57 %)
4(Very liquid)	8/36 (22.22 %)		19/58 (32.76%)
5 (Watery)	5/21 (23.81 %)		11/43 (25.58%)

Seasonal analysis

The prevalence of E. coli infection was highest in Autumn (29.41%) followed by winter (26.09%) and summer (24.59%). The infection rate was minimum in spring (16.95%) (Table III). The seasonal variations were found to be significant statistically (p<0.05).

Colostrum feeding

A total of 128 goats were given colostrum’s feeding among which 26 (20.31%) showed Salmonella cases (Table III). The results were found to be significant (P<0.05).

In the case of E. coli, 102 goats were given colostrum feeding among which 19 (18.63%) showed infection (Table III). Overall, the results were non-significant (P>0.05).

Diarrhea Duration

In the context of diarrhea duration among goats, the data indicated that goats with a one-day duration of diarrhea had a lower Salmonella (14.04%) rate while higher (26.19%) E. coli infection, whereas those with a two-day duration showed a higher rate of infection in Salmonella (20.31%) and lower rate in E. coli (19.72%) infection as compared to one-day duration. Furthermore, goats experiencing diarrhea for three or more days exhibited an infection rate of 17.72% in Salmonella and 24.44% in case of E. coli (Table III). The study revealed that there were no statistically significant associations between diarrhea duration and Salmonella infection (p>0.05) as well as between E. coli infection and diarrhea duration (p<0.05).

Body condition

Regarding body conditions, the results stated that emaciated goats showed maximum (24.39%) positive cases while good healthy goats showed 11% positive cases of Salmonella followed by lowest infection rate in fatty goats 7.84% (Table III). The results were non-significant (P>0.05). In case of E. coli infection, emaciated goats had maximum (31.91%) prevalence followed by fatty condition (18.60%) and healthy goats in good condition (14.29%) (Table III). The results were statistically significant (P<0.05). In both infections, emaciated condition had maximum prevalence.

Environmental condition

Regarding environmental condition, the data revealed that good environmental condition showed 14.04% Salmonella infection, fair environmental condition showed 18.03% positive cases while poor condition showed 19.51% infection (Table III). In this study, the results were non-significant (P>0.05). In the case of E. coli, the results revealed that fair environmental condition showed maximum (46.81%) positive cases of infection (Table III) followed by poor (19.51%) and good environmental condition (12.68%). The results were statistically significant (P<0.05).

Stagnant water

The presence of stagnant water showed 19.49% positive cases of Salmonella as compared to its absence (14.63%) (Table III). E. coli infection was also more prevalent (31.48%) in the presence of stagnant water as compared to its absence (14.13%) (Table III). The findings were significant (P<0.05) in both cases of infections.

Fecal scoring

The maximum cases (23.81%) of infection were recorded in watery feces (score 5) followed by liquid feces (score 4) and normal feces (score 1) with 22.22 and 19.05% respectively. Infection rate in semi solid feces (score 2) was 16.44%. Least number of cases for positive infection were recorded in score 3 (dysentery) with 12.24% infection rate (Table III). In case of E. coli infection, maximum positive cases were recorded in fecal score 4 (32.76%) followed by score 5 (25.58%) and score 1 (22.22%) (Table III). The results were found to be significant (P<0.05) in both infections.

DISCUSSION

Neonatal diarrhea is one of the leading causes of calf death in animal industry (Urie et al. 2018). Diarrhea and health issues in young goats pose significant challenges to the well-being of these animals, impacting the overall profitability of the goat industry (Cheng et al., 2021). It is essential to gain a deeper insight into potential biomarkers that can serve as indicators for mortality and morbidity in neonatal goats suffering from diarrhea (Dwyer et al., 2015). The current study revealed that bacterial prevalence was 43.5% in goat kids.

E. coli stands as the primary and most influential factor responsible for neonatal diarrhea in ruminants (Brunauer et al., 2021). The E. coli prevalence in this study was much lower (23.5%) than prevalence reported by previous studies in diarrheic goats; such as, 57.8% (Shabana and Enazi, 2020), 48.7% (Abd El-Tawab et al., 2020), 31.7% (Kiziltepe and Ayvazoğlu, 2022) and 27.3% (Osman et al., 2013). Mishra et al. (2018) identified 193 isolates out of 300 (64.3%). The differences in E. coli prevalence may be linked to variations in geographic distribution, calf age, weather conditions, management practices, and hygiene measures (Alam et al., 2009; Vanselow et al., 2017; Abd El-Tawab et al., 2020).

The Salmonella prevalence was 17.5% in the current study which was lower than E. coli infection. Lower rate (7%) of Salmonella infection in goats was also reported by Hawwas et al. (2022), 3.86% by Farouk et al. (2021) and 3.49% by Abd El-Twab et al. (2016). Another study reported 75.6% E. coli and 18.1% Salmonella infection in diarrheic calves (El-Seedy et al., 2016). Diagnosing salmonellosis in sheep and goats poses challenges because the clinical symptoms are not specific (OIE, 2000). Contaminated water can be considered as one of the leading causes of Salmonella infection in goat kids (Ghimire and Bhattarai, 2019).

Young goats are vulnerable to diarrhea during the initial week of their lives (Caffarena et al., 2021). In case of Salmonella infection G2 was found to be most infected. The results were in accordance with Grünberg (2020) and Duffy et al. (2010). In case of E. coli G1 was most infected age group. Moreover, the Salmonella isolation rate was highest (13.5%) between one and two years of age (Hawwas et al., 2022). E. coli K99 was found to be most common factor of diarrhea in kids in Kuwait (Majeed et al., 2018; Abdou et al., 2021) and Saudi Arabia (Shabana et al., 2017).

Male goats showed more prevalence in both infections, Salmonella (65.71%) and E. coli (31.25%). In Ethiopia, a hundred percent higher isolation rate of Salmonella was recorded in males as compared to females (0%). Saha et al. (2014) also reported higher (15.78%) prevalence in male goats as compared to females (10.71%).

The seasonal analysis revealed that Salmonella infection was highest (41.18%) during winter season followed by summer (19.12%). The percentage of infection was same in spring and autumn (13.04%). In case of E. coli infection, the prevalence was highest in Autumn (29.41%) followed by winter (26.09%) and summer (24.59%). Abdou et al. (2021) recorded more cases of E. coli diarrhea in dry season as compared to wet season.

Conclusions

Neonatal diarrhea poses a significant threat to goat mortality, leading to economic losses. A year-long research project examined Salmonella and Escherichia coli prevalence in diarrheic goat kids. Fecal samples from 200 goats under two months old were collected from various sources in the Bahawalpur district. Microbiological analysis identified bacterial presence, with higher rates in male goat kids compared to females. Local Desi breed goats had the highest prevalence. Seasonal variations were significant for Salmonella, favoring winter, while E. coli showed no significant seasonal variation.

Declarations

Funding

The study was not funded by any organization.

IRB approval

The study was approved by the IRB of University of Veterinary and Animal Sciences, No/624/DVM, dated: (16/02/2022).

Statement of conflict of interest

The authors have declared no conflict of interest.

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